DNA polymerase activity on solid support:
From diagnostics to directed enzyme evolution
Dissertation
zur Erlangung des akademischen Grades
des Doktors der Naturwissenschaften
(Dr. rer. nat.)
an der Universität Konstanz
Naturwissenschaftliche Sektion
Fachbereich Chemie
vorgelegt von
Ramon Kranaster
2009
Konstanzer Online-Publikations-System (KOPS)
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-123778
URL: http://kops.ub.uni-konstanz.de/volltexte/2010/12377/
Tag der mündlichen Prüfung: 15. Januar 2010
Prüfungsvorsitzender und mündlicher Prüfer: Herr Professor Dr. Hartig
1. Referent und mündlicher Prüfer: Herr Professor Dr. Marx
2. Referent und mündlicher Prüfer: Herr Professor Dr. Wittmann
3. Referent: Herr Professor Dr. Fischer
Teile dieser Arbeit sind veröffentlicht in:
Biotechnol J. 2010, 5(2), 224-231.
Kranaster, R., Drum, M., Engel, N., Weidmann, M., Hufert,
F.T., Marx, A.
“One-step RNA pathogen detection with reverse transcriptase
activity of a mutated thermostable Thermus aquaticus DNA
polymerase.”
EMBO J. 2010, 29(10), 1738-1747.
Obeid, S., Blatter, N., Kranaster, R., Schnur, A., Diederichs,
K., Welte, W., Marx, A., “Replication through an abasic DNA
lesion: structural basis for adenine selectivity.”
Angew. Chem. Int. Ed. 2009,
48(25), 4625-4628.
Kranaster R. & Marx A. “Taking fingerprints of DNA
polymerases: Multiplex enzyme profiling on DNA arrays”
Nucleic Acids Symposium Series, 2008,
52, 477-478.
Kranaster, R. & Marx, A. “New Strategies for DNA Polymerase
Library Screening”
ChemBioChem, 2008,
9, 694 – 697.
Kranaster, R., Ketzer, P. & Marx, A. “Mutant DNA polymerase
for improved detection of single-nucleotide variations in
microarrayed primer extension “
Chem. Eur. J., 2007;13, 6115-6122.
Kranaster, R., Marx, A. “Increased single-nucleotide
discrimination in allele-specific polymerase chain reactions
through primer probes bearing nucleobase and 2'-deoxyribose
modifications”
Chem Biol., 2007 14,185-194.
Rudinger, N.Z., Kranaster, R., Marx, A. “Hydrophobic amino
acid and single-atom substitutions increase DNA polymerase
selectivity”
weitere Publikationen:
Nucleic Acids Res., 2007; e143.
Fahrer, J., Kranaster, R., Altmeyer, M., Marx, A., Bürkle, A.
“Quantitative analysis of the binding affinity of poly(ADPribose) to specific binding proteins as a function of chain
length”
Table of contents
Table of contents
1
Introduction ........................................................................ 1
1.1
DNA Synthesis - Biological role of DNA polymerases ............................2
1.1.1 Structural model for DNA polymerase I from Thermus aquaticus .............2
1.1.2 Reaction pathway of DNA polymerases..............................................3
1.1.3 Chemical mechanism of the catalysed nucleotidyl transfer....................5
1.2
Biotechnological role of DNA polymerases..........................................6
1.2.1 Polymerase chain reaction (PCR) .....................................................6
1.2.2 Modified and mutated DNA polymerases............................................7
1.2.3 Directed evolution of DNA polymerases........................................... 10
1.2.4 Methods of mutagenesis .............................................................. 12
1.3
Accuracy of enzymatic DNA synthesis .............................................. 13
1.3.1 Chemical approach for increasing the selectivity of DNA polymerases.... 14
1.3.2 Genetic approach for increasing the selectivity of DNA polymerases...... 16
1.4
2
Aim of this work ........................................................................... 18
Results and discussion......................................................... 20
2.1
SNP detection by allele-specific real-time PCR employing chemically
modified DNA primers ............................................................................. 20
2.1.1 Introduction ............................................................................. 20
2.1.2 Results ..................................................................................... 23
2.1.3 Conclusion ................................................................................ 28
2.2
SNP detection by arrayed primer extension employing a mutated DNA
polymerase ............................................................................................ 30
2.2.1 Introduction ............................................................................. 30
2.2.2 Results ..................................................................................... 31
2.2.3 Conclusion ................................................................................ 36
2.3
Profiling of DNA polymerases by arrayed primer extension ................. 37
2.3.1 Introduction ............................................................................. 37
2.3.2 Results - Oligonucleotide-addressing enzyme assay (OAEA) ................ 37
2.3.3 Conclusion ................................................................................ 46
2.4
One-step RNA pathogen detection employing a mutated thermostable
DNA polymerase ..................................................................................... 48
I
Table of contents
2.4.1 Introduction ............................................................................. 48
2.4.2 Results ..................................................................................... 50
2.4.3 Conclusion ................................................................................ 56
2.5
Functional Studies on the Tyrosin Y671 responsible for A-rule in KlenTaq
DNA polymerase ..................................................................................... 57
2.5.1 Introduction ............................................................................. 57
2.5.2 Results ..................................................................................... 58
2.5.3 Conclusion ................................................................................ 63
3
Summary and outlook .......................................................... 65
4
Zusammenfassung und Ausblick............................................ 67
5
Materials and methods......................................................... 70
II
5.1
Reagents ..................................................................................... 70
5.2
Biochemical reagents, enzymes and kits .......................................... 72
5.3
Bacterial strains and plasmids........................................................ 72
5.4
Disposables ................................................................................. 73
5.5
Equipment................................................................................... 73
5.6
Buffers and solutions.................................................................... 76
5.7
Determination of DNA concentration............................................... 78
5.8
Oligonucleotides .......................................................................... 79
5.9
Radioactive labelling of DNA-oligonucleotides.................................. 79
5.10
Agarose gelelectrophoresis ............................................................ 79
5.11
Denaturing polyacrylamide gelelectrophoresis ................................. 79
5.12
SDS polyacrylamide gelelectrophoresis ............................................ 80
5.13
Determination of protein concentration .......................................... 80
5.14
Site directed mutagenesis ............................................................. 80
5.15
Transformation of chemically competent cells .................................. 81
5.16
DNA-sequencing........................................................................... 81
5.17
Crystal structure models ................................................................ 81
5.18
Methods for section 2.1 ................................................................. 81
Table of contents
5.18.1 Real-time PCR experiments........................................................... 81
5.18.2 Primers and templates................................................................. 82
5.18.3 DNA thermal-denaturation studies................................................. 82
5.18.4 Circular dichroism spectra ............................................................ 83
5.18.5 Kinetic single-nucleotide incorporation studies................................ 83
5.19
Methods for section 2.2 ................................................................. 84
5.19.1 Primers and templates................................................................. 84
5.19.2 Activation
of
glass
slides
and
spotting
of
amino-modified
oligonucleotides to glass slides ............................................................... 85
5.19.3 Primer extension and arrayed primer extension ................................ 85
5.20
Methods for section 2.3 ................................................................. 86
5.20.1 Primers and templates on solid support.......................................... 86
5.20.2 Primers and templates in solution experiments................................ 87
5.20.3 Overexpression of KlenTaq clones in multiwell format and cell lysate
preparation......................................................................................... 88
5.20.4 Spotting of DNA polymerase cell lysate mixtures and screening
reactions ............................................................................
88
5.20.5 Expression and purification of KlenTaq mutants................................ 89
5.20.6 Primer extension reactions in solution............................................ 89
5.20.7 Test of the influence of drying and rehydration on the KlenTaq polymerase
activity in solution................................................................................ 90
5.20.8 Spotting and immobilisation of short DNA oligomers ......................... 90
5.20.9 Estimation of surface coverage of oligonucleotides bound to the chip
surface ............................................................................................... 90
5.21
Methods for section 2.4 ................................................................. 91
5.21.1 Cloning, Protein Expression and Purification of Taq M1 Polymerase ...... 91
5.21.2 Nuclease activity assay ................................................................ 92
5.21.3 Real-time PCR, template dilution series .......................................... 92
5.21.4 Primer extension assay with an RNA template .................................. 93
5.21.5 Real time RT-PCR conditions ......................................................... 93
5.22
Methods for section 2.5 ................................................................. 93
III
Table of contents
5.22.1 Primer extension assays containing abasic site................................. 93
5.22.2 Primer extension assays containing blunt-ended DNA........................ 94
5.22.3 Site directed mutagenesis ............................................................ 94
5.22.4 Enzyme kinetics ......................................................................... 95
6
Abbreviations ..................................................................... 96
7
DNA and amino acid sequences.............................................. 98
7.1
pASK-IBA37plus ::TaqM1 nucleic acid sequence ................................ 98
7.2
TaqM1 amino acid sequence ......................................................... 100
7.3
pASK-IBA37plus ::KlenTaq wt nucleic acid sequence ........................ 101
7.4
KlenTaq wt amino acid sequence in pASK-IBA37+ ............................ 103
7.5
pGDR11 ::KlenTaq wt nucleic acid sequence .................................... 104
7.6
KlenTaq wt amino acid sequence in vector pGDR11 .......................... 107
8
References ....................................................................... 108
9
Eidesstattliche Erklärung ................................................... 117
IV
Introduction
1
Introduction
Self-replication is a process in which an entity (e.g. a cell or virus) makes a copy of
itself and is thus a fundamental basis of life. Living cells accomplish cell division by
cooperative action of a plethora of proteins and nucleic acids. Before a cell divides, it
has to replicate its own genetic information for biological inheritance. This
information is stored in a polymeric macromolecule, deoxyribonucleic acid (DNA) and
is used by all living organisms on earth. DNA consists of a chain of four different
components, the nucleotides: adenosine, cytidine, guanosine and thymidine. Each
nucleotide is a deoxyribose sugar linked with a monophosphate and a heterocyclic
base and is connected to the next base via phosphodiester linkages creating an
alternating phosphate-deoxyribose strand. Two antiparallel strands are coiled
together to form a characteristic double-helical structure (Figure 1). The phosphatedeoxyribose backbone shields the inward pointing bases, which are interacting with
the opposite DNA strand through specific hydrogen bonds forming base pairs. Adenine
pairs with thymine, and cytosine pairs with guanine (see Figure 1,(B)).
Figure 1
Double-helical structure of DNA. (A)Stick –(left) and surface model (right). PDB-code:
1BNA1. (B)Watson-Crick base pairing2 of A with T and G with C.
The sequence of the four different bases in DNA is very important because they
intrinsically carry the genetic code. A set of three neighboured bases is called a triplet
codon and usually encodes for a specific single amino acid. Thus, each specific DNA
sequence may code for at least one specific protein. The entirety of DNA in one cell
constitutes the genome, which in principal dictates the production of proteins,
enzymes, cell’s function and cell’s properties.
1
Introduction
1.1
DNA Synthesis - Biological role of DNA polymerases
In living cells, DNA synthesis is mainly divided into the processes of replication, repair
and lesion bypass. DNA polymerases are the main actors in these processes. They are
catalysing the DNA synthesis under consumption of the corresponding nucleotide
triphosphates via nucleotidyl transfer3. DNA replication is the duplication of the
genome in a semiconservative process in that each strand of the original doublestranded DNA serves as a template for the synthesis of the complementary strand. DNA
repair is the identification and repair of certain genomic DNA lesions like DNA adducts,
single and double strand breaks, photoproducts and abasic sites. DNA translesion
synthesis (TLS) is the process of lesion bypass performed by specialised DNA
polymerases able to bypass DNA lesions, which are typically strong obstructions for
these enzymes4.
1.1.1
Structural model for DNA polymerase I from Thermus aquaticus
1958 Kornberg and coworkers5 discovered the first enzymes that catalyse the
incorporation of deoxyribonucleotides into DNA extracted from Escherichia coli (E.coli)
cells. These first serendipities triggered an avalanche of DNA and RNA polymerase
studies about their biological function, properties and catalytic mechanisms.
Nowadays several crystal structures of polymerases are available in the presence and
absence of DNA, RNA and nucleotide substrates. Kim et al.6 solved the structure of
thermostable DNA polymerase I from Thermus aquaticus (Taq) by x-ray scattering
1995, which displays a typical DNA polymerase structure. The structure is very similar
to a right hand consisting of a finger domain, a thumb – and a palm domain.
Additionally, the Taq DNA polymerase has an N-terminal attached nuclease domain
(see Figure 2). Li et al.7 could further crystallise an N-terminal shortened form of the
Taq polymerase (KlenTaq) bound to the primer template DNA in the so-called 'binary
complex' and an additionally bound triphosphate constituting the 'ternary complex'.
2
Introduction
Figure 2
Crystal structure of thermostable DNA polymerase I from Thermus aquaticus (Taq). The
structure is very similar to a right hand consisting of a finger-domain, a thumb– and a palm-domain.
Additionally Taq DNA polymerase has a N-terminal attached nuclease domain. PDB-code 1TAQ7.
During catalysis, the polymerase is able to adopt two different conformations: an open
and a closed form. Especially the finger domain has to move between these two
conformations with an angle of ~46°. It is consensus that upon binding of a
triphosphate the polymerase turns into the closed conformation until incorporation of
deoxynucleotide takes place and a pyrophosphate is set free. The enzyme opens again
and may translocate along the primer template DNA or dissociates from the extended
primer template to bind to another DNA substrate.
1.1.2
Reaction pathway of DNA polymerases
The kinetic mechanism of most DNA polymerases8 can be described by a general,
simplified scheme: In the first step, the polymerase binds to the DNA substrate. The
second step involves the binding of a dNTP constituting the ternary complex. Step
three is a conformational change from the open ternary to the closed ternary complex
which is followed by the nucleotidyl transfer (step four, see also Section 1.1.3). In step
five, the enzyme releases the extended primer template DNA substrate and relaxes to
its initial conformation.
3
Introduction
Figure 3
Schematic representation of DNA polymerase catalysed DNA synthesis. The reaction
pathway is separated into eight different kinetic steps.
During step six, the pyrophosphate is released and the polymerase may continue the
synthesis on the same DNA substrate by translocation (step 7) or may dissociate from
the extended primer template to bind on another DNA substrate (step 8). The number
of added nucleotides by a polymerase during an association and dissociation at a
single DNA substrate is defined as processivity. Replicative DNA polymerases tend to be
very processive thus adding several hundred nucleotides upon binding, whereas DNA
repair involved polymerases have low processivity adding only single or a few
nucleotides. In E.coli for example, the DNA Polymerase III represents a processive DNA
polymerase responsible for DNA replication and DNA polymerase I is a low processive
representative responsible for DNA replication initiation9.
The question of the rate-limiting steps during enzymatic action has been explored by
numerous kinetic studies and with the aid of modified substrates like α-S-dNTPs10 or 2aminopurines11 in the template strand but is still discussed. In studies of the Klenow
fragment deriving from E.coli and DNA polymerase from bacteriophage T4, the
conformational change from open to close state (step 3) seems to be the rate-limiting
step11, whereas in stopped flow fluorescence studies with human DNA polymerase β
the nucleotidyl transfer step is indicated with the lowest rate constant12. Taken
together, it could be possible that DNA polymerases involved in DNA repair and/or
lesion bypass have a different rate-limiting step than DNA polymerases involved in
replication. In principle, all enzymatic steps shown in Figure 3 are reversible.
Pyrophosphorolysis is the reverse process and generates dNTPs under degradation of
4
Introduction
the primer strand. High concentrations of pyrophosphate shift the equilibrium to the
pyrophosphorolysis process, which plays an important role in drug resistance of HIV RT
polymerase13,14. Furthermore, in biotechnological applications in which incorporation
of artificial substrates into DNA by DNA polymerases are used, the degradation and
generation of native triphosphates can easily be avoided by addition of
pyrophosphatase into the reaction mix15.
1.1.3
Chemical mechanism of the catalysed nucleotidyl transfer
Although several crystal structures and numerous kinetic data have been published,
the catalytic mechanism of DNA polymerases is still discussed and not fully
understood. It is well established that a nucleophilic attack on the α-phosphorous
atom of the nucleoside triphosphate by the primer 3´C-OH group leads to formation of
the phosphodiester bond and to the release of a pyrophosphate. All known DNA and
RNA polymerases require two divalent cations (usually Mg2+) in their active center and
use a two-metal-ion mechanism for the catalysed nucleotidyl transfer. The most recent
study on the mechanism of DNA polymerases (see Figure 4) proposes an extended two
metal ion mechanism by a two proton transfer reaction catalysed by typical acid and
basic amino acids: The protonation of the pyrophosphate leaving group by a proposed
acid and the deprotonation of the 3´C-OH group by a proposed basic amino acid3.
5
Introduction
Figure 4
Extended two-metal-ion mechanism of nucleotidyl transfer which includes a general acid
catalysis3. Shown is the active center of a polymerase with a nucleoside triphosphate (red) and two
divalent metal cations (Mg2+). One metal ion is coordinated by the phosphates of the triphosphate and an
aspartate residue located in motif A of all polymerases (blue), and probably water molecules. The other
metal ion is coordinated by the 3´C-OH of the primer terminus (green), the α-phosphate of the nucleoside
triphosphate and widely conserved aspartate residues of structural motifs A and C. A proposed acid (A)
which protonates the pyrophosphate leaving group is indicated by four different model polymerases which
where used in the study of Castro et al. to proof their concept of a general acid catalysis. A proposed basic
amino acid (B) which deprotonates the 3´C-OH group stays unidentified until now. The figure was designed
according to reference3.
Both metal ions stabilise the structural adjustment. Metal ion A lowers the pKa of the
3´C-OH group and thus supports the deprotonation by an unidentified base B and the
subsequent nucleophilic attack at physiological pH conditions. It is believed that the
α-phophate adopts during this attack a SN2 type trigonal bipyramidal transition state3.
1.2
Biotechnological role of DNA polymerases
DNA polymerases are used in a plethora of biotechnological applications16. Nowadays,
they are the workhorses in numerous applications like DNA sequencing and
microarrayed nucleic acid diagnostic tools for the direct diagnosis of single-nucleotide
variations within genes, forensic DNA testing, pathogen detection, et cetera.
1.2.1
Polymerase chain reaction (PCR)
A key technology for the use of DNA polymerases in biotechnological applications is
the polymerase chain reaction (PCR), which was development by Mullis and coworkers
in 198717. In theory, PCR allows the exponential enrichment of a particular DNA
sequence by amplification of a single or few copies of template strands. For PCR
applications a DNA polymerase needs specific primers (short DNA fragments) which
contain sequences complementary to a target DNA region. During repeated cycles of
6
Introduction
heating and cooling the DNA is generated and is itself used as a template for
replication. Due to the enzymatic replication under consumption of the primers and
deoxynucleotide triphosphates (dNTPs) the selected DNA sequence flanked by the
primers is exponentially amplified. In almost every PCR application, heat-stable DNA
polymerases resistant against the thermal cycling steps necessary to physically
separate the two strands of the DNA double helix (usually at high temperatures ~95°C)
are employed. Nowadays, PCR methods in presence of fluorescent dyes (e.g.
SYBRGreenI) or fluorescence resonant probes (e.g. TaqMan)18-21 report the amount of
amplified DNA in real-time. Both fluorescent dyes and modified DNA polymerases have
significantly shortened conventional PCR methods. Consequently, real-time PCR
methods are the method of choice for the detection and quantification of DNA and RNA
targets such as retroviruses and viral pathogens21.
Today the principle of a PCR is extended in numerous biotechnological applications:
Allele-specific PCR for the detection of single nucleotide variations22-24, multiplex PCR
for the multiple amplification of different DNA fragments in one reaction vessel25,
nested PCR which increases the specificity of the DNA amplification reaction26,
quantitative PCR to quantify and compare certain DNA strands27, reverse transcription
PCR for the detection of RNA targets28 et cetera.
1.2.2
Modified and mutated DNA polymerases
Highly processive and accurate DNA polymerases are desired for cloning procedures in
order to give shorter extension times as well as a more robust and high yield
amplification. The processivity of a DNA polymerase was significantly enhanced
recently by protein fusion technology29: DNA polymerase processivity is definded as a
value of the average number of nucleotides added by a DNA polymerase per
association/disassociation with the DNA template. It is known that the Taq polymerase
(see Section 1.1.1) consists of two distinct structural and functional domains, the 5´3´ nuclease domain and the polymerase domain. The N-terminal shortened form of Taq
(KlenTaq) lacking the nuclease domain is significantly less processive than the fulllength Taq29, suggesting that the nuclease domain interacts with the DNA template
and must be involved in maintaining processivity. In 2004 Wang et al.29 impressively
demonstrated the processivity enhancement of a DNA polymerase from Thermus
Aquaticus (Taq). They fused KlenTaq and Taq with a heterologous, sequence non-
7
Introduction
specific, double-stranded DNA binding domain resulting in enzymes with increased
processivities without compromising catalytic activity and enzyme stability. By
monitoring the single primer extension products by sequencing gels, it was
demonstrated in detail that Taq wild-type added up to 35 nucleotides whereas fused
Taq produced products up to added 200 nt.
A higher DNA polymerase fidelity may increase the reliability of diagnostic application
systems30. Marx and coworkers31 demonstrated that the selectivity of Taq DNA
polymerase can be increased by nonpolar substitution mutations of three amino acids
QVH (Gln, Val, His) of motif C directly neighboured to the catalytic center.
Furthermore, they showed that these obtained mutants can be applied as a useful tool
in genotyping assays like allele specific real-time PCR31,32.
To enhance the efficiency of forensic DNA testing, DNA polymerases resistant to
inhibitors from blood and soil would enable the PCR without prior DNA purification.
Barnes and coworkers33 have recently evolved Taq DNA polymerase mutants with
enhanced resistance to various known inhibitors of PCR reactions, including whole
blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as
well as high concentrations of DNA binding dyes. The mutated position of the Taq
polymerase (Glu 708) is located in an alpha-helix region on the surface of the enzyme,
known as “P-domain” (residues 704–717). This domain is situated about 40 residues
apart from the “finger” domain, which binds the incoming dNTPs and interacts with the
single stranded DNA template. Because the mutation site is at the hinge region one
might speculate that it may affect the movement of the finger domains during
incorporation. The described mutation in this example is not directly involved in
interaction between substrate and enzyme thus indicating a so-called remote effect34.
The recovery of ancient DNA samples, which could be more than 40 000 years old,
requires DNA polymerases with an increased substrate spectrum to efficiently amplify
and overcome typical DNA lesions35. In 2007 Marx and coworkers36, and Holliger and
coworkers35 published successfully evolved DNA polymerases that are able to amplify
from highly damaged DNA templates and bypass lesions found in ancient DNA such as
abasic sites.
Further improvements of DNA polymerases are required, for example, to meet the
requirements of next generation DNA sequencing technologies, which rely on the
ability of DNA polymerases to efficiently process modified nucleotides37. For example
8
Introduction
the sequencing technology from Illumina Inc. uses fluorescent reversible terminator
deoxyribonucleotides38. The triphosphates have a 3’-O-azidomethyl group, which
stops the polymerase after incorporation of one nucleoside. All four 2’deoxynucleoside triphosphates (A, C, G and T) are additionally labelled with a different
removable fluorophore to determine the sequence by fluorescence readout after each
incorporation step.
Figure 5
Highly modified deoxyribonucleotides are used in the sequencing technology from
Illumina Inc. The triphosphate (A) has to be efficiently processed by the DNA polymerase. The next
sequencing cycle can begin, after chemical removal of the fluorescent dye and 3´-OH protecting group (B).
To improve the efficient incorporation of these unnatural nucleotides they had to engineer the active site of
9°N DNA polymerase. The figure was designed according to reference 38.
After readout, the 3´-O-azidomethyl group and the fluorescent dye will be chemically
removed by tris(2-carboxyethyl)phosphine (TCEP), that the next single incorporation
cycle can begin. However, to improve the efficient incorporation of these unnatural
nucleotides they had to engineer the active site of 9°N DNA polymerase to gain a
sufficient sequencing setup.
Taken together, customized and artificially engineered DNA polymerases that lead to
more robust and specific reaction systems are urgently needed.
9
Introduction
1.2.3
Directed evolution of DNA polymerases
Native proteins and enzymes are the natural products of several million years of
evolution. Alliances of enzymes in one living organism cause it to be good or less good
adapted to certain environmental conditions. Natural selection takes place in a way
that the best adapted to the given conditions prevails.
This process can be artificially enhanced by modern biochemical methods in order to
obtain enzymes, e.g. DNA polymerases, with new features. Alterations are mainly
achieved by directed molecular evolution using genetic complementation and/or
screening28,31,32,36,39-43, phage display44-47, or in vitro compartmentalization48-50. In
general, three steps are required for a successful directed molecular evolution of DNA
polymerases: introduction of mutations by certain methods of mutagenesis,
expression of different enzyme variants and screening or selection of best enzyme
variants. These steps can be repeated until the desired feature is obtained (see Figure
6).
Figure 6
Scheme of directed evolution of DNA polymerases. 1. Introduction of arbitrary mutations
indicated as red dots. 2 Separation of different mutants. 3. Screening/Selection of mutants by appropriate
assays. These steps can be repeated until the desired feature is obtained e.g. higher mismatch
discrimination.
After creation of a mutant library, a mutant separation process is needed which also
ensures a linkage between a specific enzyme genotype and the respective phenotype.
Selection or screening approaches are described in the literature for the directed
10
Introduction
evolution of DNA polymerases. Common selection methods are phage-display47,
compartmentalised self replication35 (CSR) or reporter plasmid assays43.
For example, Vichier-Guerre et al.47 employed phage display to select DNA polymerase
mutants with about two orders of magnitude higher catalytic efficiency for reverse
transcription when compared with the natural enzyme. In phage-display, the DNA
polymerases are expressed and displayed together with a substrate on the surface of a
phage. The polymerase mutant displayed onto the phage particle has to convert a
linked substrate into desired product, which is then selected for example by affinity
chromatography. One disadvantage might be the high degree of cross-reactions
between a polymerase on one phage and a substrate attached to another phage.
Holliger and coworkers49 employed CSR to evolve polymerases that can extend
mismatches and common lesions found in ancient DNA. They demonstrated that these
engineered polymerases could expand the recovery of genetic information from
Pleistocene specimens35. For a CSR method, each polymerase gene is encapsulated in a
compartment formed by a heat-stable water-in-oil emulsion. Each polymerase has to
replicate its own encoding gene and therefore results in a very high adaptive burden
depending on the specific selection system.
Loeb and coworkers43 employed a reporter plasmid assay for the selection of DNA
polymerase I mutants from E.coli with increased fidelity. In a reporter plasmid assay, a
plasmid is used which contains a reporter gene for example containing an antibiotic
resistance gene but with an opal codon. Selection of mutants is possible by comparing
the reversion frequencies of the wild-type with the mutants.
In standard screening methods, the mutants must be separately expressed in multiwell plates so that the phenotype is directly connected to the corresponding mutant in
each well. Subsequent screening reactions of mutants can be processed by either
primer extension reactions or PCR. Real-time PCR screening yields a very high
sensitivity due to the exponential enrichment of the product32. Barnes and coworkers
for example employed a radioactive labelled nucleotide incorporation assay to screen
successfully for DNA polymerase mutants, which are more resistant against common
inhibitors present in blood and soil samples33.
Until now, all screening methods for DNA polymerase mutants are restricted to a single
reaction or a single modified substrate such as a non-natural base or a mismatched
primer/template situation. In contrast to that, microarrays should offer the ability to
11
Introduction
screen in a multiplexed and parallel manner for several different reactions and new
functions. This approach was followed in Section 2.3.
1.2.4
Methods of mutagenesis
Numerous strategies and methods of mutagenesis can be found in the literature:
Site directed mutagenesis is a good strategy when proper information about structural
and substrate-enzyme interactions is available. The mutation sites are rationally
designed and are introduced by site directed mutagenesis protocols using mutagenesis
primers carrying the respective nucleic acid sequence for the desired mutation51.
Special enzyme features may change simply by introducing these point mutations. This
strategy could be a good starting point for further directed evolution.
Saturated mutagenesis offers another option to test one amino acid position with all
native possible amino acid substitutions. The amino acid position can be randomised
using degenerated mutagenesis primers. When a single codon is randomised, the
library size can be small (3-4 hundreds mutants) and leads to 99.9% probability of
having all possible mutations included52.
Arbitrary gene mutations can be introduced by error-prone PCR28,53 (epPCR). In epPCR
high magnesium or manganese concentrations and/or imbalanced mixtures of
deoxynucleotide triphosphates during the PCR are used, causing the DNA polymerase
to produce incorporation errors. Unfortunately this encompass a few disadvantages:
Due to the nature of template amplification, an early occurring mutation in the first
cycles of PCR might be enriched during amplification and thus overrepresented in the
resulting protein library54. Additionally, DNA polymerases used in epPCR preferentially
produce transition than transversions errors due to the steric demands of similar bases
(purines A, G and pyrimidines T, C). At least certain amino acid exchanges do occur
very infrequently, because exchange of one nucleotide is not enough to change a
whole amino acid codon and mutation of two neighboured nucleotides is not occurring
very often55. Nevertheless, error-prone PCR creates a good initially library for directed
evolution methods especially when few information on enzyme structure and
important amino acid residues exists.
Furthermore, arbitrary gene mutations can be introduced by other techniques such as
sequence saturation mutagenesis56 (SeSaM). This method uses gene fragmentation by
iodine cleavage of previously introduced phosphorothioate groups randomly
12
Introduction
distributed in the gene. After fragmentation, these single stranded DNA fragments are
used as primers for the following full-length gene synthesis. During this step, artificial
bases with universally binding properties are used to ensure arbitrary randomisation.
Disadvantages of this method are that DNA fragments smaller than ~70 nt are not
mutagenized due to the employed DNA extraction and purification procedures56. The
independence from the mutational bias of DNA polymerases using epPCR is exchanged
for the different base-pairing preferences of universal or degenerated bases.
Homologous recombination methods enable the shuffling of different mutations and
additionally introduce new mutations as well. In the DNA shuffling method,
homologue genes are fragmented by DNase digestion and afterwards reassembled to
the full-length gene by PCR57. An alternative method is the staggered extension
process (StEP) which also allows the combination of different homologous genes58. It
uses highly abbreviated annealing and extension steps during PCR to generate
staggered DNA fragments. This procedure promotes crossover events along the full
length of the template sequences resulting in a library of chimeric polynucleotide
sequences.
1.3
Accuracy of enzymatic DNA synthesis
Due to its complementary structure, DNA can be copied using the respective DNA
strand as a template. The energy differences based on hydrogen bonding between a
correct Watson-Crick base pair and an incorrect one are not very high (~ 1-3 kcal/mol)
and result theoretically in a high error-rate of one per 100 incorporated
nucleotides59,60. In living cells during replication, DNA polymerases have to copy the
whole genome, in human cells these are more than 6 billion nucleotides61. The
genomic sequence would rapidly undergo changes after each cell division. Fortunately,
nature has evolved polymerases with error-rates much lower than one would expect
from thermodynamic considerations62,63. In bacteria for example, the overall accuracy
of DNA synthesis reaches one error of 108-1010 incorporated nucleotides62,64. Even in
eukaryotic cells error-rates >1010 are reached8. The impact of incorrectly incorporated
nucleotides can vary: On the one hand, mutations in the genetic sequence may be a
silent mutation without any effect or may lead to better-adapted organisms; on the
other hand, mutations may just as well lead to development of cancer or cell death.
13
Introduction
Misincorporation rates by DNA polymerases in E.coli are determined by 103 – 106,
depending of course on the misincorporated base, indicating enzymatic processes that
increase the accuracy beyond thermodynamic limitations65. Factors like proofreading
and mismatch repair by base and nucleotide excision repair (BER and NER) improve
these rates further to the overall error-rates mentioned above (108-1010).
Kool and coworkers showed that the efficient and selective enzymatic DNA synthesis is
not dependent on the hydrogen bonding between Watson-Crick base pairs alone66. In
detail, they studied base analogues that lack the ability for hydrogen bonds, but still
have similar size and shape compared to the natural bases66 (see Figure 7, A). DNA
polymerases were still able to use these analogues as substrates in a selective manner.
The base analogues were selectively incorporated opposite of the respective correct
template base and furthermore used as a template base selectively addressing for the
correct triphosphate.
Figure 7
(A) Representative isosteric base analogue N in comparison with nature base cytosin C.
(B) Pyrene base opposite an stable abasic site analogue.
Along these lines, they constructed a pyrene instead of a natural base bearing
triphosphate, which has similar size and shape as a full base pair. This artificial
triphosphate was preferentially incorporated opposite of an abasic site instead of a
nucleobase67 (see Figure 7, B). All these findings lead to the assumption that size and
shape of the incoming dNTP play an important role for DNA polymerase selectivity.
Further experiments concerning structural studies68,69 have led to the common
understanding that the geometry of the DNA base pair is regulated by a close fit in the
polymerase active site70.
1.3.1
Chemical approach for increasing the selectivity of DNA polymerases
Incorporation of an incorrect nucleotide results in the forming of a mismatch and leads
to an altered geometry within the DNA duplex. DNA polymerases are able to sense
14
Introduction
these mismatches and display significantly reduced extension rates compared to a
matched base pair situation22,71,72 (see Figure 8).
Figure 8
Matched and mismatched primer template situation. DNA polymerases are able to
differentiate between these cases resulting in reduced mismatch extension rates compared to matched
base pair situations. Black bars represent primer/template duplexes.
One approach to enhance DNA polymerase ability in discriminating between matched
and mismatched situations is to use 3´ chemically modified primer probes.
Latorra et al. introduced locked nucleic acid (LNA) modification at the 3'end of primer
probes and demonstrated that this modification leads to an increased single
nucleotide discrimination in allele-specific PCRs73. Along this line, Gaster et al.24,74
introduced several 4'-C-modifications at the 3´primer end and could show that these
modifications in combination with Vent (exo-) reveal highly increased single
nucleotide discrimination properties. Especially a polar 4´-C-methoxymethylen group
showed superior discrimination properties between matched and mismatched primer
template situations. Presumably one might hold thermodynamic reasons responsible
for this effect, but thermal denaturation studies and CD spectra revealed that
differences between stabilities of canonical over non canonical duplexes at the
3´terminal primer end are negligible22,24. Thus, it is unlikely that this effect derives
from differential duplex stabilities of 4'C-modified versus unmodified duplexes. It is
more likely that increased steric constraints and slightly disturbed geometries in the
active center especially in the case of mismatched substrates contribute to the
selectivity of the outcome of this process. Marx and coworkers75,76 could show that
altering the steric demand of the DNA substrate results in a more selective polymerase
regarding the discrimination of single nucleotide variations. Additionally, these
beneficial properties of modified primer probes were independent of buffer conditions
and applicable in different sequence contexts24. Taken together the employment of
primer probes bearing modifications at the 3’ end allowed increasing the selectivity of
asPCR significantly. These primer probes showed increased discrimination properties
15
Introduction
between matched and mismatched cases in the asPCR as well as in primer extension
and arrayed primer extension systems19,74,77.
1.3.2
Genetic approach for increasing the selectivity of DNA polymerases
Intensive crystal structure explorations revealed that DNA polymerases have subtle
hydrogen-bonding networks, especially with the minor groove of the primer/template
substrates78,79. It is believed that they have direct impact on the DNA polymerase
selectivity. On the one hand, it could be possible to alter the acceptor potential of the
DNA substrate. This approach was partially followed during my diploma thesis by
substituting the carbonyl functionalities in thymidine for thiocarbonyl groups.
Thiolated thymidines have decreased H-bonding abilities compared to the native
carbonyl groups and could have a significant effect on the selectivity of DNA
polymerases which is described and analysed in Section 2.1. In addition, other
modifications like electron withdrawing groups are imaginable but are accompanied all
with cost-intensive consumption of chemically modified primer probes.
On the other hand, it might be more effective to have DNA polymerases with improved
single nucleotide discrimination properties in combination with unmodified primer
strands and thus obviate the need for chemical modifications of the primer probes.
Loeb and coworkers selected DNA polymerase I mutants from E.coli showing enhanced
accuracy in DNA synthesis43. Mutants were selected by using a reporter plasmid
harbouring an antibiotic resistance gene. One double mutant (K601I, A726V)
exhibited a ten-fold enhanced accuracy without a significant reduction in activity43.
The mutation A726V is located in the M-helix, which is at the juncture of the fingers
and palm subdomains. It lacks any direct contacts with the substrates but may disrupt
the function of the hinge and may give more time for kinetic proofreading. The K601I
mutation is located near motif 1 which is involved in the binding and positioning of
the DNA substrate80.
Summerer et al.31 focused on three residues within the motif C of proofreadingdeficient DNA polymerases I from Thermus Aquaticus. motif C is well-known for having
hydrogen bonds with the DNA substrate via the minor groove and is directly
neighboured to the catalytic center of the DNA polymerase. They found that mainly
unpolar amino acid substitutions lead to increased single nucleotide discrimination
properties. Along these lines, Rudinger et al.72 could transfer this concept to DNA
16
Introduction
polymerases from the family B and constructed a double mutant of the DNA
polymerase from Pyrococcus furiosus (Pfu). This mutant is ten fold more selective for
the discrimination of unmodified mismatched primer templates and may build the
basis for further enhancements regarding methods for the detection of single
nucleotide variations (see also Section 2.22.2). Until now, fidelity mechanisms of DNA
polymerases are not fully understood. Taken together mutations, which decrease or
delete distinct polar interaction with the DNA substrates, may generally result in
enzymes with higher selectivity. Unfortunately, it seems that mutations with very high
impact on DNA polymerase selectivity appear in tandem with a significant reduction of
enzymatic activity.
17
Aim of this work
1.4
Aim of this work
The aim of this work was the functional analysis and recruitment of mutated DNA
polymerases for improved biotechnological applications.
Strerath et al.
23
demonstrated that DNA polymerases have increased mismatch
discrimination features under the presence of 4´C modified triphosphates or primers.
It is believed that a subtle hydrogen-bonding network of the enzyme with minor
groove of the primer/template substrate contributes to selectivity of a DNA
polymerase78,79. To proof this concept, small thiomodifications at the 2- and 4-position
on thymidine were introduced and tested regarding their effects on DNA polymerase
selectivity.
In this context a new diagnostic application for the detection of single nucleotide
variations, under recruitment of DNA polymerases by arrayed primer extensions,
should be established. It was previously demonstrated by Summerer et al.31 that
certain mutations in DNA polymerases lead to increased single nucleotide
discrimination properties. Along these lines, Rudinger et al.72 showed that a double
mutant of the DNA polymerase from Pyrococcus furiosus (Pfu) is ten fold more selective
for the discrimination of unmodified mismatched primer templates. These features of
the double mutant should be exploited in establishing a reliable arrayed method for
the detection of single nucleotide variations.
Besides investigating DNA polymerase selectivity, a new arrayed approach for the
functional profiling of DNA modifying enzymes should be established enabling
multiplexed profiling of several enzyme features in high-throughput. The system
should be based on the spatial separation of different covalently attached DNA
substrates on a glass slide and their selective addressing by template oligonucleotide
hybridisation.
Besides screening of DNA polymerase features on solid support, other functional
mutations and changes at DNA polymerases should be tested to study their effect on
enzyme activity and behaviour. In detail, Sauter et al.28 evolved an N-terminal
shortened form of the DNA polymerase from Thermus aquaticus (Taq) with highly
increased reverse transcriptase activity. The attachment of a 5´-3´ nuclease domain
constituting the full-length Taq enzyme should be performed and the functionality of
18
Aim of this work
both combined activities should be tested. The resulting enzyme should be further
evaluated regarding its usefulness for one-step RNA detection systems.
At least single mutations in the active center of a DNA polymerase should be tested to
obtain more insights concerning non-templated DNA synthesis catalysed by some DNA
polymerases following the A-rule. In this context dNTP incorporation opposite abasic
DNA lesions and at blunt-end primer template complexes should be tested and further
evaluated in kinetic incorporation studies.
19
Results and discussion
2
Results and discussion
2.1
SNP detection by allele-specific real-time PCR employing
chemically modified DNA primers
2.1.1
Introduction
Within the human genome comprising approximately 3 billion nucleobase pairs,
individuals differ in approximately 0.1 percent of the nucleotide sequence61,81. The
most frequent among these sequence variations are single-nucleotide polymorphisms
(SNPs) which are changes in a single base at a specific position in the genome82-84.
Figure 9
Representative single nucleotide variation at a specific position in the genome
SNPs are defined as sites in which the less common variant has a frequency of at least
1% in a population. A direct linkage exists between some of these dissimilarities and
certain diseases. Additionally, different effects of drugs on different patients can also
be linked to SNPs82,84,85. Moreover, SNPs can be used to determine someone’s ancestry.
Recently, it has been shown that SNPs mirror the European landscape in a way that
regional provenance may be estimated with an accuracy of 100km86,87. Thus,
considerable efforts have focussed on finding new SNPs and elucidating connections
between them and certain phenotypes. On average, each human carries three to four
million SNPs which can be detected with a variety of methods, at least by whole
genome sequencing technologies38. In a first step, high-throughput sequencing
methods that identify unknown nucleotide variations are needed. This is followed by
an investigation of the medicinal relevance of these variations. After assessment of the
exact sequence context other methods are needed for high-throughput screening of
populations in the search for known SNPs or to analyse individuals for SNP patterns.
Obviously in the latter case methods are essential that allow for time- and costeffective verification of distinct nucleotide variations in daily laboratory practice19,8895
. Many methods rely on amplification of the target gene before analysis can take
20
Results and discussion
place. Thus, methods that enable amplification and analysis in a single step are
desirable.
In principle, allele-specific PCR (asPCR) comprises these features96-103. The concept of
asPCR is based on the principle that a DNA polymerase catalyses DNA synthesis from a
matched 3’-primer terminus, while a mismatch due to hybridisation of the primer
probe to a different sequence variant should obviate DNA synthesis and, therefore DNA
amplification should be abolished (see Figure 10).
Figure 10
In principle, the DNA polymerase catalyses the DNA-synthesis in the match case
(upper half); due to incorrect hybridisation at the primer end the DNA synthesis should be oppressed in the
mismatch case (lower half) (Black bars represent primer/template duplexes).
This conceptually simple and straightforward procedure is hampered somehow by the
sequence dependency of the selectivity99-103. Thus, identification of the appropriate
reaction conditions requires tedious optimisation. In earlier publications, it has been
shown that an essential improvement may be gained in this method by employing
chemically modified primer probes23,24,73,76,104,105. The employment of primer probes
bearing a 4’-C-modification at the 3’ end allowed the selectivity of asPCR to be
increased significantly. These primer probes showed increased discrimination
properties between matched and mismatched cases in the asPCR, as well as in primer
extension and arrayed primer extension systems74. Additionally, these beneficial
properties of modified primer probes were independent of buffer conditions and
applicable in different sequence contexts23,24,73,76,104,105. Especially the combination of
primer probes bearing 4’-C-methoxymethylene residues at the 3’ end together with the
commercially available DNA polymerase from Thermococcus litoralis (Vent (exo-) DNA
polymerase) was found to exhibit superior performance in allele discrimination24 (see
Figure 11).
21
Results and discussion
Figure 11
4´-C-methoxymethylen residues at the 3´terminal position of primer probes
lead to increased allele specific discrimination using Vent (exo-) DNA polymerase. (Black bars represent
primer/template duplexes, red T represents a 4´-C-methoxymethylen residue at the 3´end of a primer)
Encouraged by these findings the effects of some nucleobase modifications in
conjunction with the previously described 4’-C-methoxymethylenated 2’-deoxyribose
residues were studied with regard to discrimination in the asPCR. In order to modify
the size and hydrogen-bonding ability of the substrates, the carbonyl functionalities in
thymidine were substituted for thiocarbonyl groups. Thiolated thymidines have an
enlarged steric demand, due to the 0.45 Å longer double bond length106 and
additionally decreased hydrogen-bonding abilities compared to the native carbonyl
groups (see Figure 12).
Figure 12
(A) Exchange of Oxygen O with Sulphur S at position 2 (2ST) and position 4
4S
( T); (B) Connolly surface with electrostatic charge distribution. Substitution of the carbonylgroup with
thiocarbonylgroup leads to an enlarged steric demand, due to the 0.45 Â longer double bond-length106 and
to a decreased H-bonding ability compared to the native carbonyl group.
To explore the effects of 2-S and 4-S substitutions in conjunction with 4’-C
modification on the allele-discrimination in PCRs, the respective modified
oligonucleotides were successfully synthesised during my diploma thesis in
2005/2006107. In that work, the 2-S and 4-S thiolated commercially available
nucleosides and the synthesised (4-S/2-S)-4’-C-methoxymethylene thymidines were
coupled to a solid long-chain amino alkyl modified controlled pore glass (LCAA-CPG)
support according to established protocols108. The solid supports were subsequently
22
Results and discussion
transferred into suitable cartridges and employed in standard automated DNA
synthesis to yield the desired oligonucleotides 4ST, 2ST, TOMe, 2STOMe, 4STOMe.
Thereupon, modified oligonucleotides were used in allele-specific real-time PCRs. For
proof of principle experiments, it was decided to use a PCR template (90nt long) within
the human acid ceramidase and the Farber disease109 sequence context. Two reactions
were conducted at a time in parallel: One PCR was conducted by employing a template
bearing a deoxyadenosine (dA) residue opposite the respective 3’-terminal thymidine
in the primer probe. In the other experiment the same primer probe was combined
with a template strand that had the same sequence apart from a dA to deoxyguanosine
(dG) mutation opposite the 3’-terminal thymidine moiety (see Figure 10). An
unmodified reverse primer was used for both setups. PCRs were analysed by employing
real-time double-stranded DNA detection through SybrGreen I fluorescence by using
appropriate thermocycler equipment110. First results during my diploma thesis
indicated that thiolated thymidines, especially
2S
T,
2S OMe
T
and
4S OMe
T
, may increase
single-nucleotide discrimination by DNA polymerase.
2.1.2
Results
After successful synthesis of the base and/or 4´C modified oligonucleotides (4ST, 2ST,
TOMe,
2S
TOMe,
4S OMe
T
) and first preliminary asPCRs during my diploma thesis107, the
previous results obtained were repeated and further analysed in more detail:
The threshold-crossing point (Ct) as a measure for amplification efficiency was
determined for each primer probe respectively. This parameter is defined as the point
in which the reporter’s fluorescence (SybrGreen I) exceeds the background
fluorescence significantly and crosses a threshold.
23
Results and discussion
Figure 13
Results of real-time asPCR experiments obtained by using primer probes bearing
4’-C-methoxymethylene residues at the 3’ end in combination with nucleobase thiomodifications. Results
are shown with primer probes 2ST, 4ST, TOMe, 2STOMe, 4STOMe or the unmodified primer T as indicated. PCR
amplification in the presence of target template A (solid line) or template G (dashed line) is shown. All
experiments were conducted under the same conditions (see the Experimental Section).
The difference in the threshold-crossing points (ΔCt) of canonical (A-T/T*) versus noncanonical (G-T/T*) primer–template amplification is a measure for single-nucleotide
discrimination. Amplification efficiency and the ability to discriminate against singlenucleotide mismatches varied with the modification employed (see Figure 13, Table 1).
Table 1
Analyses of realtime asPCRs. Comparison of ΔCt-values obtained from base and 4´-Cmethoxymethylene modified primer probes.
5´-...AGGA T/T*
(primer probe, 20nt)
3´-...T CC T N TCCA.. (template, 90nt)
Ct (N=A)
ΔCt
5
0-0.5
2S
5
3
4S
T
5
1
TOMe
6
9
2S OMe
6
12
4S OMe
20
19
T
T
T
T
T*=modified thymidine 2ST, 4ST, TOMe, 2STOMe or 4STOMe
N= A or G, respectively
24
Results and discussion
Usage of the
2S
T probe introduced some degree of selectivity (2.5-3 PCR cycles) as
compared to use of the unmodified probe. Strikingly, these effects were significantly
enhanced when a 4’-C-methoxymethylene modification was present in conjunction
with a 2-thiolation.
2S OMe
T
had superior properties when compared with the
unmodified T, 2ST and TOMe probes. Different results were obtained when the effects of
4-thiolation on asPCRs were investigated. The 4ST probe had no significant effect on
the amplification selectivity or the efficiency. When 4STOMe was used, the amplification
efficiency decreased significantly, as indicated by the Ct value of 20. Interestingly, this
was combined with an unprecedented high ΔCt value of 19 cycles. Agarose gel analysis
of the resulting reaction products revealed that, when mismatched primer–template
complexes containing the
4S OMe
T
probe were used, thermocycling resulted in
amplification of non-specific PCR products.
To gain further insights into the origin of the observed effects, thermal-denaturing
studies were conducted and CD spectra were recorded. Duplexes between the
respective primer strands (T,
2S
T,
4S
T, TOMe,
2S OMe
T
and
4S OMe
T
) and 33-mer templates
corresponding to matched (A–T/T*) and mismatched cases (G–T/T*) resulted in nearly
superimposable CD spectra. This result indicated little, if any, dependence of the
overall helix conformation on the presence of a matched or mismatched case and
modifications at the nucleobase and/or 2’-deoxyribose (see Figure 14).
25
Results and discussion
Figure 14
Resulting Circular Dichroism spectra from respective primer strands (T, 2ST, 4ST,
OMe 2S OMe
4S OMe
T , T and T , 20nt) hybridised to templates (33nt) corresponding to matched (A–T/T*) and
mismatched cases (G–T/T*). (A) T match, T mismatch, 2ST match, 2ST mismatch, 4ST match and 4ST
mismatch; (B) TOMe match, TOMe mismatch, 2STOMe match, 2STOMe mismatch, 4STOMe match and 4STOMe mismatch.
T*= modified thymidines 2ST, 4ST, TOMe, 2STOMe or 4STOMe as described above.
Next, thermal-denaturing studies were conducted in order to investigate the impact of
a chemical modification at the 3’-terminus on duplex stability. Only small variations in
melting behaviour were found (see Table 2).
Table 2
Melting temperatures Tm [°C] derived from thermal denaturing experiments of primer
probes in matched (A-T/T*) and mismatched (G-T/T*) complexes with respective template strand. All
experiments were conducted with same DNA and buffer concentrations.
5´-...AGGA T/T*
(primer probe, 20 nt)
3´-...T CC T N TCCA..
(template, 33nt)
T
matched case (A-T/T*)
mismatched case (G-T/T*)
65.5
64.5
2S
66.1
66.3
4S
T
67.5
67.4
TOMe
65.5
66.0
2S OMe
66.3
65.0
4S OMe
67.0
66.1
T
T
T
T*=modified thymidine 2ST, 4ST, TOMe, 2STOMe or 4STOMe
N=A or G, respectively
26
Results and discussion
Interestingly, all duplexes containing modified strands had slightly increased melting
points when compared to the native duplexes, in both matched and mismatched cases.
Thus, the influence of a single nucleotide at the primer terminus that has been
modified at the 4’-C-deoxyribose and/or the nucleobase on the intrinsic formation of
aberrant conformations or duplex stability is small.
Steady-state extension efficiencies were measured to quantify the influence of the
depicted chemical modifications at the primer probe 3´end on the enzyme action.
Following kinetic constants were determined using single-nucleotide incorporation
experiments under single-completed-hit and steady-state conditions, as previously
described111: kcat= first order rate of catalysis; KM= Michaelis constant and kcat/KM=
incorporation efficiency (see Table 3).
Table 3
Kinetic analyses of single nucleotide insertions of matched (A-T/T*) and mismatched (GT/T*) primer termini performed by Vent (exo-) DNA polymerase. Incorporation of dATP was observed using
the modified primer probes 2ST, 4ST, TOMe, 2STOMe and 4STOMe in comparison with unmodified T primer pro be.
5´-...AGGA T/T*
(primer probe, 20 nt)
3´-...T CC T N TCCA..
(template, 33nt)
matched case (A-T/T*)
mismatched case (G-T/T*)
KM
kcat
kcat/KM
KM
kcat
kcat/KM
[μM]
[min-1]
[min-1μM-1]
[μM]
[min-1]
[min-1μM-1]
0.23±0.02
1.11±0.1
4.8
51.8±6.0 0.48±0.05
0.009
2S
0.26±0.06
0.85±0.04
3.3
89.7±30
0.18±0.01
0.002
4S
T
0.26±0.08
1.09±0.21
4.2
33.7±9.2 0.48±0.03
0.014
TOMe
33.6±4.2
0.14±0.02
0.004
n.a.
n.a.
n.a.
2S OMe
30.2±1.3
0.21±0.02
0.007
n.a.
n.a.
n.a.
4S OMe
26.5±2.4
0.10±0.01
0.004
n.a.
n.a.
n.a.
primer
T
T
T
T
T*=modified thymidine 2ST, 4ST, TOMe, 2STOMe or 4STOMe
N=A or G, respectively
n.a.=not accessible; no nucleotide insertions were observed when up to 8nM of DNA
Polymerase, up to 1h incubation time and up to 600 μM of dATP (higher dATP
concentrations caused inhibition of the reaction) were applied.
27
Results and discussion
The best discrimination properties of the non-sugar-modified primer probes were
shown by
2S
T, which is coherent with the results obtained in real-time asPCR
experiments (described above). Recently, similar results were obtained for the
incorporation of the respective thiolated thymidine triphosphates (TTPs)112. The
increased selectivity of the system comprising 2ST is mainly achieved by a decreased
steady-state kcat value in the mismatched case, in comparison to that of the
unmodified system. One can envision that DNA amplification from matched versus
mismatched DNA complexes under PCR conditions (that is, 200 μM deoxynucleoside
triphosphates (dNTPs)) very much depends on the kcat value of the proceeding
nucleotide incorporation. In real-time PCR, the signal generation is dependent on the
formation of double stranded DNA. Thus, the reduced steady-state kcat value of the 2STcomprising system in the mismatched case might well be the cause of the aboveobserved single-nucleotide discrimination ability in allele-specific PCR. 4’-Cmodification has significant effects on the extension efficiency. The efficiency is
greatly diminished due to a significantly increased KM value (about 100-fold) and an
approximately 5- to 10-fold decreased kcat value. This is in line with earlier findings,
although different enzymes and modifications were used75. However, when singlecompleted hit conditions were used (>10-fold excess primer/template over DNA
polymerase concentration), no extension of mismatched primer termini was observed.
As demonstrated by our results, these limitations can be overcome in PCRs in which
standard dNTP concentrations are employed that are higher than the measured KM
values, thereby resulting in an efficient PCR when matched primer/templates are
employed.
2.1.3
Conclusion
Taken together, it was shown that primer probes that bear thiolated thymidines are
able to increase single-nucleotide discrimination in allele-specific PCRs. These
modifications, either at the 4’-C-deoxyribose or on the nucleobase of a singlemodified nucleotide at the primer terminus, do not have any significant effect on
duplex stability and the conformation of the respective primer–template complex.
Therefore, the characterised discrimination properties must result from the specific
interaction between the DNA polymerase, the template–primer probe duplex and the
28
Results and discussion
incoming dNTPs. Nucleobase thiolation in conjunction with 4’-C-methoxymethylene
modification at the 2’-deoxyribose exhibits the most pronounced effects. These
compounds are readily available and can be incorporated into DNA strands by using
standard oligonucleotide chemistry. This real-time PCR system supersedes recently
discovered approaches24,32 that use unmodified nucleobases. The described system
with the 2STOMe primer probe can be useful for the direct diagnosis of single-nucleotide
variations within genes, such as single-nucleotide polymorphisms or point mutations,
directly without the need of further time- and cost-intensive post-PCR analysis.
These results were published in R. Kranaster and A. Marx, Chem. Eur. J., 2007, 13,
6115-6122.
29
Results and discussion
2.2
SNP detection by arrayed primer extension employing a
mutated DNA polymerase
2.2.1
Introduction
The vast majority of genomic alteration events is based on single nucleotides113. Single
nucleotide polymorphisms (SNPs) are changes in a single base at a specific position in
the genome. These changes are differentiated from point mutations in their frequency
(>1%) in a population and are occurring on average approximately every 1.000-2.000
nucleotides in the human DNA114. Thus, each human carries 3-4 millions SNPs and they
can lead to modified structures, activities and functions of expressed proteins, if they
are located in coding and regulating gene-regions. Hence, these variations in our
genetic make-up are closely associated with complex disorders such as cancer,
diabetes, vascular diseases, some forms of mental illness, and are known to be major
players in an individual’s predisposition to side effects of drugs61,82,83. SNPs were
recently used for the genetic differentiation between subpopulations. The existing
differences were characterized by a strong correlation between geographic and genetic
distances and lead to a genetic map which mirrors the geography within Europe quite
well86,87.
Many methods for the detection of nucleotide variations are described19,92,115-119. The
single nucleotide variations are commonly discriminated by sequence-specific
hybridisation or dye-labelled nucleotide incorporation, ligation or invasive cleavage.
These systems exploit small differences, e.g. in thermodynamic stability and
electrostatic interactions as well as within enzyme recognition processes for obtaining
allele-specific properties19,92,115-119. Enzymatic approaches can enhance the
discrimination of sequence variants beyond what can be achieved by hybridisationapproaches alone120. A spatially addressable allele discriminating probe layout e.g.
DNA microarrays represents a promising method for multiplexed detection of SNPs.
Microarrays are highly efficient, parallel, and contain the opportunity for medicinal
diagnostics in a high throughput manner in the future.
In previous publications it has been shown that a reliable and enhanced single
nucleotide discrimination via primer extension or PCR can be achieved and increased
30
Results and discussion
by chemical modification of the 3´- terminal nucleotide of the primer probe, which
binds opposite the respective SNP site of a DNA template22-24,74,76,104 (see also Section
2.1.)
Marx and coworkers discovered DNA polymerase mutants that exhibit significantly
increased selectivity in extending a matched primer template duplex versus the
mismatched counterpart31,32. Along these lines Rudinger et al. created one DNA
polymerase mutant of Pyrococcus furiosus (Pfu). It contains the two amino acid
exchanges D541L/K593M (henceforth called M2) and was designed by substituting two
polar amino acid residues by hydrophobic amino acids with as close as possible steric
demands72. Rudinger et al. could demonstrate that M2 exhibits increased primer
extension selectivity in homogeneous assays, especially in allele-specific real time
PCRs. Thus, the concept of allele-specific DNA synthesis from the solution phase was
transferred to a solid support. A primer extension assay in microarray format was used
as an allele-specific sensor employing the selectivity-increased DNA polymerase
mutant M2.
2.2.2
Results
Signal generation in microarray formats relies on the incorporation of a signal e.g. a
fluorophor. Thus, in order to validate whether the mutated DNA polymerase M2 is
capable of incorporating a dye-labelled nucleotide, primer extension reactions were
conducted. A commercially available fluorophore labelled dUTP analogue was
investigated (F3-dUTP, a Rhodamine-B derivative attached to dUTP) as surrogate of
the native TTPs. It was found, that a
32
P-5’-end labelled primer is elongated by the
wild-type enzyme as well as by M2 to yield the full-length product in the presence of all
four natural dNTPs (Figure 15, lanes 1).
31
Results and discussion
Figure 15
Radioactive primer extension reactions to access the incorporation of F3-dUTP
by wild-type (wt) DNA polymerase (left panel) and M2 (right panel). A Section of the primer template is
shown on the figure. M: Marker, reaction without enzyme; Lanes 1: reaction with all 4 native dNTPs; Lanes
2: primer extension reactions with dCTP, dATP, dGTP and F3-dUTP; Lanes 3 primer extension with all 4 dNTPs
and 7.5% substitution of TTP by F3-dUTP. All reactions were conducted under same conditions and
incubation times.
The wild-type enzyme added an additional nucleotide in a nontemplated manner as it
has been observed for 3’–5’-exonuclease-deficient DNA polymerases before121.
Interestingly, M2 produced the blunt-ended 35nt full length product without
significant formation of longer products. When the natural TTP was substituted for F3dUTP, a reaction product was formed that migrated slower than the original band
deriving from experiments comprising TTP (lanes 2) in denaturing polyacrylamide gel
electrophoresis. The retardation can well be explained by the additional size of the
fluorophore. Similar effects have been reported before122-125. Noteworthy, the
respective bands were fluorescent when read-out of the dried gel was performed using
a fluorescence imager (data not shown). Furthermore, it could be shown that F3-dUTP
is able to compete with TTP incorporation since even in the presence of only 7.5% F3dUTP and of 92.5% TTP the slower migrating band was detectable after analysis (see
Figure 15, lanes 3).
In the next step, the concept of fluorophor incorporation based primer extension was
transferred onto solid support. For that purpose primer probes were immobilised on
1,4-phenylene diisothiocyanate (PDITC) -activated glass slides via aminoalkyl linkage
at their 5´-termini74 and comparative primer extension reactions with wild-type Pfu
32
Results and discussion
DNA polymerase as well as with M2 were performed. A schematic illustration is given in
Figure 16.
Figure 16
Schematic illustration of allele-specific primer extension reactions. Primer
probes are covalently attached onto an aminopropyl PDITC activated glass substrate. In a matched case
(left hand, T-A) primer extension reactions are expected to take place by Pfu DNA polymerase promoted
DNA synthesis, whereby in a mismatched case (right hand, C-A) the primer extension reaction should be
obviated. A fluorescence signal is generated by partial incorporation of F3-dUTP instead of dTTP.
At first, experiments were conducted in the sequence context of human acid
ceramidase comprising the transition mutation A107G which is involved in the onset of
Farber disease109. For immobilisation, the relevant 5´-terminal aminohexyl modified
oligonucleotide (20nt) was spotted as nine replicate blocks onto the PDITC activated
glass slides. To study the match to mismatch ratio the respective primer probe blocks
with a respective 3’-terminal nucleobase thymine (T) or cytosine (C) were spotted
directly neighbouring each other (see Figure 17). The ratio between the two
fluorescence intensities (primer probe T / primer probe C) derived after primer
extension reactions in the presence of F3-dUTP and readout was defined as the
discrimination ratio and should be directly dependent on the degree of allele-specific
discrimination. Both enzymes were able to show sufficient primer extension reaction
and incorporation of the fluorescent-labelled F3-dUTP on the surface. The primer
elongation reaction with wild-type Pfu DNA polymerase resulted only in poor
discrimination properties with match (T-probe) to mismatch (C probe) ratios of 4:1.
However, using M2 the discrimination ratio increased 4-fold to 17:1(see Figure 17).
33
Results and discussion
Figure 17
Allele-specific primer extension in microarray format with Farber A template.
The microarray spotting design is shown on the right. Farber primer probes with 3´terminal T or C were
spotted in 3x3 blocks directly neighbouring each other. Resulting microarray data from wild-type and
mutant Pfu DNA polymerase are shown in the middle. Fluorescence values between primer probe T and
primer probe C. Arrayed primer extension reactions were carried out with wild-type enzyme and M2. All
reactions were conducted under equal conditions on the same slide with the identical amount of enzyme,
template and dNTPs.
Next, it was investigated whether the ability of the microarray system to discriminate
between single nucleotide variations could be applied to other sequence contexts in a
selective and multiplexed manner. Therefore, additional to the Farber sequence
context, two single nucleotide variations that are of considerable medicinal interest
were chosen. The factor V Leiden G1691A mutation is believed to be responsible for a
genetic predisposition to thrombosis126,127. A mutation G735A in the human
dihydropyrimidine dehydrogenase (DPyD) gene leads to reduced activity of this
enzyme and treatment with the anticancer drug 5-fluorouracil (5-FU) results in fatal
haematopoietic, neurological, and gastrointestinal intoxication since the mutated
enzyme is inefficient in inactivating 5-FU128. All possible six different primer probes
were spotted (see Figure 18) and primer extension reactions were conducted in the
presence of one, two, or three of the respective templates (see Figure 18, Figure 19).
34
Results and discussion
Figure 18
Microarray spotting design of three different sequence contexts (Farber, Leiden
and DPyD) and two different 3´primer termini T or C for generating a match- and a mismatch-case. The
resulting fluorescence image after microarrayed primer extension reaction with Leiden A template as a
representative experiment. Primer extension reactions were carried out with wild-type enzyme and with M2.
All reactions were conducted under equal conditions on the same glass slide with the same amount of
enzyme, template and dNTPs.
In case of the wild-type enzyme, only poor discrimination ratios (2:1 – 5:1) were
obtained due to errant extension of non-cognate primer probes. Using the M2 enzyme
the specific template showed primer extension predominantly with increased
discrimination ratios (14:1 – 20:1) which were in the same range found in the first
studies of the Farber sequence context (compare Figure 17 and Table 4). No significant
fluorescence was detected at locations where non-cognate primer probes were
spotted.
Figure 19
Resulting fluorescence images after microarrayed primer extension reaction
under presence of depicted DNA templates using mutated Pfu DNA polymerase M2 [yx].
35
Results and discussion
Table 4
Discrimination ratios of Pfu DNA polymerase wild-type and M2 within three different
sequence contexts. F: Fluorescence intensities (primer probe T / primer probe C) after primer extension
reaction under presence of F3-dUTP in arbitrary units. Error was calculated from standard deviations of
replicates and error propagations from the averaged calculated ratios (Fmatch/Fmismatch).
Sequence context
wild-type
M2
Fmatch/Fmismatch
Fmatch/Fmismatch
Farber
2.5 ± 0.2
14 ± 2
Leiden
1.5 ± 0.5
14 ± 1
DPyD
4.6 ± 0.3
20 ± 3
2.2.3
Conclusion
Taken together, it has been demonstrated that in arrayed primer extensions the Pfu
DNA polymerase mutant M2 is more accurate than the corresponding wild-type
enzyme. Recently, it was reported that single nucleotide discrimination via primer
extension or PCR can be achieved and increased by chemical modification of the 3´terminal nucleotide of the primer probe22-24,74,76,104 (see also Section 2.1). It is shown
that mutated DNA polymerases in combination with unmodified primer strands are
able to fulfil the same demands on solid support and thus obviate the need for
chemical modifications of the primer probes. The entire microarray experiment
starting from the primer extension reaction (20 min reaction time), including washing
steps and readout of the microarray, was finished within 40 minutes without further
optimization of parameters. The system might be enhanced by optimizing reaction
buffer composition and washing conditions. In addition, the two-fold lower activity of
the depicted Pfu DNA polymerase mutant M2 compared to the wild-type enzyme72
might be improved by further mutation. Nevertheless, the accuracy and simplicity of
the demonstrated approach using a mutated DNA polymerase and unmodified DNA
primer probes was demonstrated. The system depicted herein could provide the basis
for further advancements in microarrayed nucleic acid diagnostics.
These results were published in R. Kranaster, P. Ketzer and A. Marx, ChemBioChem,
2008, 9, 694 – 697.
36
Results and discussion
2.3
Profiling of DNA polymerases by arrayed primer extension
2.3.1
Introduction
DNA polymerases are used in a plethora of biotechnical applications, especially in the
polymerase chain reaction (PCR), genetic cloning procedures, genome sequencing,
and diagnostic methods16. Highly processive and accurate DNA polymerases are
desired for cloning procedures in order to give shorter extension times as well as more
robust and high yield amplification. A higher DNA polymerase fidelity may increase the
reliability of genome sequencing and diagnostic systems30. Amplification of ancient
DNA samples requires DNA polymerases with an increased substrate spectrum to
efficiently overcome typical DNA lesions35. To enhance the efficiency of forensic DNA
testing, DNA polymerases resistant to inhibitors from blood and soil allow PCR without
prior DNA purification33. Further improvements of DNA polymerases are required, for
example, to meet the requirements of real-time DNA single-molecule sequencing,
which relies on the ability of DNA polymerases to efficiently process modified
nucleotides37. Overall, customized and artificially engineered DNA polymerases that
lead to more robust and specific reaction systems are urgently needed. Directed
evolution holds promise for engineering nucleic acid polymerases with altered
properties129,130. Alterations are mainly achieved by directed molecular evolution using
genetic complementation and/or screening28,31,32,36,39-43, phage display44-47, or in vitro
compartmentalization48-50. All reported methods for DNA polymerase evolution are
restricted to a single enzyme property, for example, increased selectivity or the ability
to efficiently process DNA lesions28,31,39-44,46,50. Therefore, a microarrayed device was
developed to overcome these obvious limitations that allow the multiplexed screening
of several enzyme features in parallel.
2.3.2
Results - Oligonucleotide-addressing enzyme assay (OAEA)
First, I had to establish a new arrayed screening system, which is based on the spatial
separation of different covalently attached DNA substrates on a glass slide and their
selective addressing by oligonucleotide hybridization. This setup allows simultaneous
and multiplexed profiling of several enzyme features with high throughput. It has to
37
Results and discussion
be mentioned that standard microarray equipment was sufficient to conduct and
quantify the reactions. The developed method is time and cost efficient, and requires
only minimal amounts of reagents (see Materials and methods).
The principle behind this approach, termed oligonucleotide-addressing enzyme assay
(OAEA), is depicted in Figure 20.
Figure 20
Principle of oligonucleotide-addressing enzyme assay (OAEA). Short colored
bars represent immobilized primer strands; long colored bars represent templates, which are selectively
addressed by hybridization with the complementary immobilized primer strands. Green stars represent
streptavidin–Alexafluor546 conjugates which bind to incorporated biotin on the extended primer strands.
It mainly consists of two spotting steps and an incubation step. In the first step,
5’-NH2-(CH2)6-modified DNA oligonucleotides are covalently attached in defined rows
of
spots
on
phenylenediisothiocyanate-activated
glass
slides74,77.
These
oligonucleotides act as primer strands in the DNA polymerase catalyzed reactions.
Noteworthy, the slides can be stored at 4°C for several weeks without compromising
their usability. Then in a second spotting step, enzyme mutants suspended in a buffer,
which contains the respective DNA templates, dNTPs and a biotin-dUTP derivative are
applied. The mixtures are distributed in nanoliter quantities at the same positions as
the previously spotted primers. During this procedure, the spots dry out.
Subsequently, the glass slides are incubated in a humidity chamber (see Figure 21) at
50°C, which causes the spots to rehydrate.
38
Results and discussion
Figure 21
Picture of a humidity chamber with 4 representative glass slides. A glass petridish (25 x 150 mm) is filled with water and equipped with a microarray pedestal (white). For incubation
the humidity chamber is standing in a heating furnace at 50°C.
It was found that drying and rehydration had little effect on the activity of the
employed DNA polymerase (see below) after independent studies in solution (see
Figure 22). In detail, a dilution series of purified wild-type KlenTaq (wt) (400, 40, 4,
0.4, 0.04, 0.004 nM, respectively) was freeze dried and rehydrated afterwards in the
original amount of pure water. Subsequently, all aliquots of KlenTaq wt were incubated
in primer extension reactions in solution. No significant differences could be observed
between dried and rehydrated samples compared to unaffected samples.
Figure 22
Drying and rehydration effects on primer extension activity of KlenTaq DNA
polymerase. Dilution series of purified KlenTaq wt (400, 40, 4, 0.4, 0.04, 0.004 nM, respectively) without
drying (left) and after drying and rehydration (right). M: Marker, reaction mix without enzyme
Rehydration of the spots creates separate reaction entities in which the respective DNA
polymerase mutant is expected to process the primer–template duplexes covered by
39
Results and discussion
the respective spot. All reactions on the glass slide are stopped by repeated rinsing
with an aqueous detergent solution. In case of primer extension, biotin-dUTP will be
incorporated in the respective extended primer strand. A fluorescence signal is
generated by incubation with a streptavidin–Alexafluor546 conjugate, which binds to
the incorporated biotin (Figure 23).
Figure 23
Schematic illustration of arrayed primer extension reactions. Primer probes are
attached covalently on aminopropyl PDITC activated glass slide. After spotting of enzyme the reaction mix
with biotin labeled dUTP and incubation in a humidity chamber, a fluorescence signal is generated by
incubation with a streptavidin–Alexafluor546 conjugate, which binds to the incorporated biotin at the
extended primer strand.
To estimate the loading density and the amount of attached DNA primers on the
surface, the resulting fluorescence signal of biotinylated immobilised primers were
compared with a directly spotted streptavidin-alexa fluor®546 conjugate: a
3´-biotinylated and 5´-aminohexyl modified DNA oligomer was immobilised in
replicates on a freshly activated aminosilane slide as described in Section 5.19.2. After
incubation with a streptavidin-alexa fluor®546 solution, a dilution series of
streptavidin-alexa fluor®546 (1μM – 30nM, 0.4 nl) was spotted on the same glasslide
directly neighboured to the DNA replicates. Without any additional washing the whole
slide was scanned using a GenePix microarray scanner machine. Under the assumption
that the binding stoichiometry between a streptavidin conjugate to the immobilised
biotinylated DNA strand is 1:1, a linear regression of the fluorescence values obtained
from the dilution series allowed for calculation of the amount of immobilised DNA. The
40
Results and discussion
amount of immobilized DNA could be estimated to be about 100 amol and of each
enzyme to be 200–300 amol (for more details see Section 5.20.9).
To evaluate this approach it was investigated whether the depicted microarray system
is able to distinguish between active and non-active DNA polymerase mutants. Thus, at
first primer strands were spotted onto the slide, that bind to a template (120 nt) in a
fully matched fashion. For screening purposes a library of an N-terminal shortened
form of the DNA polymerase from Thermus aquaticus (KlenTaq) obtained by errorprone PCR28 was used131. The polymerase library was expressed in E. coli cells
distributed in 96-well plates. After expression, cell lysis and heat denaturation of the
host proteins, the crude lysates were mixed with a buffer containing DNA templates,
dNTPs, and a biotin-dUTP derivative. Subsequently, the polymerase-containing
solutions were spotted in a way that each enzyme variant covered two of each set of
primer spots. Thus, duplicated results under identical conditions were obtained.
After incubation, reaction termination and rinsing, the slides were treated with a
solution containing a streptavidin–alexafluor546 conjugate, rinsed again and
quantified using a standard microarray reader. It was also tested whether fluorescently
labelled 2’-deoxynucleoside- 5’triphosphates (see F3-dUTP, Section 2.2.2) could be
used instead of the biotin streptavidine-based approach. Interestingly, only high
fluorescence background values were obtained resulting from unspecific binding of the
modified triphosphate even after extensive washing conditions.
Next, ten mutants identified as non-active, and ten mutants, identified as active by
the formation of fluorescent spots on the microarray were randomly chosen for further
characterization (see Figure 24).
41
Results and discussion
Figure 24
OAEA evaluation.a) Partial sequence of primer and template used in this
experiment. b) Ten randomly chosen non-active mutants (1–10) and ten randomly chosen active mutants
(a–j). c) Denaturing polyacrylamide gel electrophoresis analysis of primer extensions performed in solution
by the mutants depicted in (a). M: marker, reaction without cell lysate.
Indeed, in solution the OAEA non-active mutants showed only little primer extension
in contrast to the OAEA active mutants, which yielded the full-length product (38 nt,
Figure 24c). A shorter template was used for primer extension reactions in solution
which was in the same sequence context as the one used in screening in order to
obtain better resolution in product analysis by gel electrophoresis. Again the enzymes
add an additional nucleotide in nontemplated manner as it has before been observed
for 3’-5’-exonucleasedeficient DNA polymerases (also see Figure 15)121. Thus, the
findings in the solution phase are in excellent agreement with the results obtained on
the solid phase.
Next, the simultaneous processing of five different primer–template duplexes was
investigated by a library of DNA polymerase mutants. A template harbouring an abasic
site analogue was employed, as abasic sites are known to hinder numerous DNA
polymerases4,132. Also several different substrates that are mismatched at the 3’-end of
the primer including a single terminal mismatch, a single distal mismatch and one
triple mismatch duplex were tested (for detailed DNA sequences see Figure 25 below
and Section 5.20.1).
42
Results and discussion
Figure 25
Partial sequences used in this study.
As a reference, a non-modified fully matched primer–template complex was used.
Without further optimization, a small library of 736 KlenTaq mutants was screened
with these five primer–template duplexes. Each enzyme-containing entity was spotted
with all five primer–template duplexes in duplicate. Generally, for the fully matched
case the highest fluorescence intensity was found as it was expected. The signal-tonoise ratio of wild-type (wt) KlenTaq with the non-modified primer–template substrate
was consistently higher than 35:1, whereas for a negative control reaction (n.c.) using
a bacterial extract without the KlenTaq gene, no fluorescence was detected (see Figure
26).
Figure 26
a) OAEA results derived from cell lysates expressing the KlenTaq wild-type (wt)
gene and a negative control (n.c.) from cell lysates harboring a plasmid without the KlenTaq gene. b)
Fluorescence intensity profiles (F.I.) along the red arrows as indicated in (a).
This signal-to-noise ratio exceeds values reported previously in fluorescent-based
screening approaches36,133. Extension activities for the other duplex systems like the
43
Results and discussion
templates with the basic site, the single terminal mismatch and the single distal
mismatch were characteristically decreased to values between 15–30% of the matched
case, while for the triple mismatch case values of 3–5% were observed. A broad
spectrum of different activities was found within the screened 736 enzyme mutants.
That makes it possible to profile and classify DNA polymerases by specific activity
“fingerprints”. The most interesting three mutants (m1, m2 and m3) were overexpressed, purified by Ni-NTA affinity chromatography and further characterized in
primer extension reactions in solution.
Figure 27
SDS-PAGE of purified KlenTaq DNA polymerase mutants (m1-3) and wild-type
(wt) enzyme. Equal concentration (coomassie staining).
Mutant m1 showed extraordinary discrimination against mismatches within the primer
template construct and showed lower bypass activity for the substrate with the abasic
site than the wild-type enzyme did (Figure 28).
44
Results and discussion
Figure 28
Evaluation of mutants with altered properties identified by OAEA. Processing of
various DNA substrates in solution by evolved KlenTaq mutants (m1–m3) in comparison to the wild-type
enzyme. All reactions were performed with identical reaction buffer and dNTP concentrations (100 mm
each). Enzyme concentrations and incubation times: for matched, terminal and distal mismatched: 1 nm,
15 min; for triple mismatched and abasic site template: 50 nm, 60 min. In the latter two cases higher
enzyme concentrations and prolonged incubation times were required to promote extension of the more
aberrant DNA complexes. For more experimental details see Materials and Methods. M: marker, reaction
without enzyme.
Interestingly, mutants m2 and m3 showed the opposite behavior and were more
efficient at bypassing the abasic site (located at nucleotide position 24) than the wildtype enzyme. Mutants m2 and m3 also showed higher activity in extension reactions
with the mismatched than that displayed by either the wild-type enzyme or m1 is
observed.
Sequencing of the mutated genes revealed only a single mutation for m1, namely
serine 460 (S460) to proline (P). Interestingly, S460 is located in helix H of the thumb
domain of KlenTaq (Figure 29). As a result of the known helix-breaking ability of
proline134, it can be assumed that the H helix has lost its conformation in the S460P
mutant.
45
Results and discussion
Figure 29
Mutations in the evolved KlenTaq DNA polymerases m1 (red), m2 (green) and
m3 (blue) are mapped on a ribbon representation of KlenTaq (PDB code:1QSS135). The inset highlights the
H helix with the observed mutation sites from m1–m3.
Two mutations were found for m2 (Y455N, V766A) and four mutations for m3 (L359P,
R457G, E537G, V586I). Currently it is not known which amino acid exchange
contributes most to the observed effects. Nevertheless, interestingly in both the m2
and m3 mutants, as well as the m1 mutant, mutations in the H helix are observed
(Figure 29). The H helix does not have direct van der Waals contact with the DNA
substrate, but the findings show that mutations at this helix are able to influence the
processing of the various substrates. With this screening approach “fingerprints” of
DNA polymerases can be taken by direct comparison of their properties in processing
different substrates. These findings indicate that the ability of lesion bypass in m2 and
m3 is linked to their lower discrimination against mismatches. This is in accord with
previously reported results36,49. For m1 it appears that this enzyme in general is more
discriminatory against aberrant DNA structures such as mismatched primer ends and
DNA lesions.
2.3.3
Conclusion
In summary, the herein described method is a new microarray-based approach for DNA
polymerase evolution, termed oligonucleotide-addressing enzyme assay (OAEA). First
experiments have proven the practicability of this approach and new DNA polymerase
46
Results and discussion
mutants with altered properties could be identified. In comparison with other known
directed evolution approaches for DNA polymerases, OAEA offers several significant
advantages. This approach allows the multiplex detection of various DNA polymerase
activities in parallel under identical conditions. In addition, in OAEA each reaction can
be duplicated readily. These features render OAEA reliable and less prone to falsepositives and false-negatives. Furthermore, all steps can be performed by automated
pipetting devices, allowing high-throughput analysis requiring only minuscule
amounts of reagents. Given the recent advances in microarray fabrication with more
than 6 000 000 possible discrete features136 on one chip, the depicted assay can be
extended for the simultaneous ultrahigh-throughput multiplexed screening of
extensive libraries with thousands of mutants. Furthermore, other possible
applications can be foreseen. As all reactions are separately addressable by single
enzyme entities, the method allows for parallel profiling of DNA polymerases from
different origins. Additionally, other DNA-modifying enzymes like ligases and
endonucleases can be included in multiplex directed evolution approaches using
OAEA.
These results were published in R. Kranaster and A. Marx, Angew. Chem. Int. Ed., 2009,
48, 4625 – 4628.
47
Results and discussion
2.4
One-step RNA pathogen detection employing a mutated
thermostable DNA polymerase
2.4.1
Introduction
Emerging pathogenic viruses like Hantaviruses137 or the recently occurring Influenza A
virus subtype H1N1138 can develop into a public health risk. To monitor and detect
their appearance and circulation, reliable rapid pathogen detection methods are
needed. Apart from several antibody based assays139 like the hemagglutination
inhibition test (HI), enzyme immunoassay (EIA) and virus neutralization tests (VN),
nucleic acid detection assays (NA) such as the polymerase chain reaction (PCR) are
among the most reliable detection techniques used for pathogen detection17. For PCR
a DNA polymerase needs specific primers (short DNA fragments) with sequences
complementary to a target DNA region. During repeated cycles of heating and cooling
new DNA is generated and is itself used as a template for replication. Due to the
enzymatic replication under consumption of the primers and deoxynucleotide
triphosphates (dNTPs), the selected DNA sequence framed by the primers is
exponentially amplified in theory. Almost in every PCR application heat-stable DNA
polymerases are employed which remain active during the thermal cycling steps
necessary to separate the two strands of the DNA double helix (usually at high
temperatures of ~95°C). Nowadays real-time PCR methods in presence of fluorescent
dyes e.g. SYBRgreen I or TaqMan probes18-21 report the amount of amplified DNA in
real-time and have significantly shortened conventional PCR methods. Hence, they are
the method of choice for detection and quantification of DNA and RNA targets such as
retroviruses and viral pathogens21. In routine virological diagnostics probe based real
time PCR systems are state of the art.
Two enzymes are needed to detect RNA by a reverse transcription (RT)-PCR. In a first
crucial step for RT-PCR, the RNA target is reverse transcribed into the complementary
DNA strand. This is performed by an efficient non-thermostable RNA dependent DNA
polymerase (reverse transcriptase) followed by real-time amplification of the
transcribed target by a thermostable DNA polymerase21. In real time RT-PCR,
fluorescent probes are used to increase the level of specificity and to avoid detection
48
Results and discussion
of non-specific side-products18,19,140: The fluorescent probe hybridises to a sequence
in-between the flanking primer sequences of the PCR target. A fluorophor and a
quencher molecule are covalently attached to the 5’ and 3’ end of the TaqMan probe
allowing for Förster resonance energy transfer (FRET) to occur between both
molecules, resulting in suppressed fluorescence of the fluorophor dye. During the PCR
extension steps, a DNA polymerase, which harbours an active 5´-3´nuclease domain,
degrades the DNA stretch of a fluorescence probe that is annealed to the target strand.
The fluorophor molecule is cleaved from the probe and released from close proximity
to the quencher molecule, resulting in increased fluorescence. Thus, the generated
fluorescence signal is directly proportional to the amplified target molecules after
each cycle. The most critical step in this method is the conversion from the RNA target
into DNA. This reverse transcription is prone to failure, because RNA can host highly
stable secondary structure motifs such as hairpins and G-quadruplexes which
complicate or even prevent reverse transcription141. Thus, thermostable reverse
transcriptases, which are able to work at higher temperatures, are of urgent need, to
increase reliability and sensitivity of RNA pathogen detection systems. It is known that
some DNA polymerases for example from Thermus aquaticus exhibit a low intrinsic RT
activity that is too inefficient for a fast and reliable RT PCR based RNA detection142.
Myers and Gelfand141 reported a DNA polymerase from Thermus thermophilus (Tth)
that exhibits increased reverse transcriptase (RT) activity exclusively in the presence of
Mn2+ ions, but unfortunately for many biotechnological applications like pathogen
detection or gene expression analysis employment of Mn2+ is inappropriate21.
Sauter el al.28 previously described an evolved N-terminal shortened form of DNA
polymerase from Thermus aquaticus (KlenTaq) which was obtained by directed
evolution and has increased reverse transcriptase activity. Presented here is the
cloning and characterisation of a respective full-length Taq DNA polymerase mutant
(henceforth called Taq M1) including the 5´-3´ nuclease domain which is required for
the well established real-time detection by TaqMan probes. The successful addition of
the nuclease activity under conservation of the previously evolved reverse
transcriptase activity is demonstrated. The results show that Taq M1 has the same PCR
sensitivity and the same 5´-3´nuclease activity as the respective wild-type Taq DNA
polymerase under perpetuation of the increased reverse transcriptase ability. Finally,
49
Results and discussion
RT PCR studies demonstrate the usefulness of Taq M1 for fast and reliable RNA
pathogen detection
2.4.2
Results
Sauter et al.28 previously discovered by directed enzyme evolution a thermostable DNA
polymerase (KlenTaq M1) with RT PCR activity. An overview of the mutations in the
KlenTaq domain (dark blue) is shown in Figure 30 on a ribbon representation of the
crystal structure7. Here, by using the scaffold of KlenTaq M1, a full length Taq DNA
polymerase (Taq M1) was constructed with the respective amino acid mutations of the
KlenTaq M1.
Figure 30
Taq M1 mutations are mapped on a ribbon representation of Taq DNA
polymerase (PDB code 1TAQ7). KlenTaq domain is coloured in deep blue and 5´-3´-nuclease domain in
light blue.
Taq M1 was over-expressed in E. coli cells and purified by Ni-NTA affinity
chromatography followed by a gel filtration (see Figure 31).
50
Results and discussion
Figure 31
SDS-PAGE gel of purified Taq DNA polymerases Taq wt and Taq M1.
First, it was tested if the mutations of the KlenTaq M1 domain influence the activity of
the added N-terminal attached 5´-3´nuclease domain. Therefore, a stable DNA hairpin
structure was used to which a radioactive labelled cleavage substrate anneals at the
complementary site (Figure 32, (A)).
Figure 32
5´-3´-nuclease activity.
(A) Hairpin structure of the template and the 22 nt
substrate (bold). The arrow indicates the expected cleavage position based on reported studies on E. coli
DNA polymerase I and Taq DNA polymerase143. (B) Separated reaction products by denaturing PAGE. (C)
Product formation (quantified ratio of product to the sum of product and substrate) after certain periods
(0, 5, 15, 30, 60 min).
This structure harbours a displaced 5’ end and a frayed 3’ primer terminus and has
been shown to be the preferred substrate for cleavage by the 5´-3´nucleases of Taq
DNA polymerase and E. coli DNA polymerase I143. Figure 32 shows the time dependent
cleavage of the 22 nt long substrate resulting in the cleaved shorter product. Taq M1
51
Results and discussion
exhibits similar nuclease activity than the wild-type Taq DNA polymerase (Taq wt).
Thus, it appears that the mutations in the polymerase domain have little if any effect
on the nuclease activity. Next, the PCR activity of Taq M1 compared to Taq wt was
investigated (Figure 33). A 100 nt long DNA template which was diluted tenfold
stepwise from 1 nM to 10 fM concentration of template was amplified.
Figure 33
PCR activity test of Taq wt compared to Taq M1.
(A) and (B) Real-time
PCR curves of a template dilutions series using Taq wt (A) and Taq M1 (B) including a negative control
without template (dashed line). Generally, all reactions were performed in triplicates. (C) Ct values vs.
detected DNA template molecules.
The resulting real-time PCR amplification curves using SYBRgreen I were measured and
are shown in Figure 33. By comparing the threshold-crossing points (Ct) between Taq
wt and Taq M1, very similar Ct values and thus same PCR sensitivity as for Taq wt
(Figure 33,(C)) was found.
To investigate the ability of Taq M1 for reverse-transcription (RT) in comparison with
Taq wt, primer extension reactions were conducted using a RNA template strand. A
20 nt 5´-32P-phosphate labelled DNA primer strand was annealed to its complementary
site on a 30 nt RNA template strand. As a control reaction the respective DNA template
was used. Furthermore, reactions were conducted at different temperatures ranging
from 60-72°C to find an optimal RT temperature. After 10 minutes of incubation the
reaction products were analysed by denaturing PAGE. The control reaction in the
presence of the respective DNA template yielded with both employed enzymes the
expected 31 nt long full-length product (Figure 34, C = control reaction). Both
52
Results and discussion
enzymes added an additional nucleotide (31nt) in non-templated manner as it has
been observed for 3’-5’-exonuclease deficient DNA polymerases before77,121,144. On the
contrary, in reactions employing the RNA template the wild-type enzyme (Taq wt)
extended the primer by seven nucleotides and only at temperatures below 65°C.
Surprisingly, at higher reaction temperatures no extension products were visible at all.
Whereas using the same reaction conditions, mutant M1 reverse transcribed the RNA
template significantly more efficiently and produced the full-length product.
Interestingly, the reverse transcription efficiency was significantly reduced at
temperatures higher than 70°C.
Figure 34
Reverse transcription primer extension of Taq M1 compared to Taq wt under
equal reaction conditions. M = Marker, reaction mix without enzyme. C = control reaction with the
corresponding DNA template. Incubation (10 min, 10 nM enzyme concentration) was carried out at
different temperatures ranging from 60-72°C (from left to right: 60.1, 60.3, 61.2, 62.5, 63.9, 65.3, 66.7,
68.1, 69.5, 70.8, 71.7, 72.0°C).
Next, real-time RT PCR experiments were performed employing the 3569 base pair long
RNA genome from bacteriophage MS2, which was diluted stepwise from 10 nM to
10 fM. In the experimental set-up a 100 nt RNA target-sequence had to be first reverse
transcribed within 30 min and amplified subsequently according to a standard onestep RT-PCR protocol (Figure 35). It was found that only the mutated Taq M1 is able to
efficiently use the RNA target. In contrast Taq wt showed only low PCR activities and
amplified the reverse transcribed RNA target with Ct value differences of more than
~10 cycles depending on the RNA template concentration.
53
Results and discussion
Figure 35
Real-time RT PCR activity test of Taq wt compared to Taq M1.
(A) and (B) Real-time RT PCR curves of a tenfold template dilutions series with Taq wt (A) and Taq M1 (B)
including a negative control without template (dashed line). Generally, reactions were performed in
triplicates. (C) Ct values vs. number of detected RNA template molecules.
These are expected results according to previous findings of a low intrinsic RT activity
of Taq DNA polymerase141,142. Inspired by the finding of the temperature dependence
of an RT reaction (see Figure 34) real-time RT PCR experiments were conducted at
different temperatures (ranging from 60-72°C) during the RT step (see Figure 36) and
a clear temperature dependence of the RT was found which is in good agreement to the
previously conducted RNA primer extensions (Figure 34).
Figure 36
Temperature dependence of Taq M1 reverse transcriptase activity. Resulting Ct
values of subsequent amplification vs. applied RT temperature. RT reaction (15 min incubation, 5 nM
enzyme concentration) was carried out at different temperatures ranging from 60-72°C.
The RT optimum reaction temperature with the lowest Ct value is between 63-68°C. The
efficiency drops drastically when the RT temperature is below 63°C or higher than
54
Results and discussion
70°C. After these promising results next these findings were validated in the detection
of pathogenic RNA obtained from natural sources.
To test the performance of the newly evolved TaqM1 enzyme in an established realtime RT-PCR TaqMan assay, the enzyme was send to Dr. Manfred Weidmann from the
University Medical Center Göttingen, Department of Virology.
Dr. Weidmann and coworkers tested the enzyme for its use in an assay for the detection
of Dobrava virus145 which is the causative agent of hemorrhagic fever with renal
syndrome (HFRS) in Europe and Asia and hantavirus pulmonary syndrome in America.
The real time RT-PCR assay for the Dobrava virus has an analytical sensitivity of 102
molecules when using the Roche Kit containing an aptamer blocked Tth polymerase.
The TaqM1 enzyme did not perform well in the real-time RT-PCR assay using the TrisHCl (NH4)2SO4-buffer (pH 9.2) used in the previous real-time PCR and the extension
assays. TaqM1 performed much better at an analytical sensitivity of 103 molecules
detected in a less basic 50 mM bicine buffer (pH 8.2) (see Figure 37).
Figure 37
Dobrava virus detection by one step real-time RT PCR. Crossing points (CP) are plotted
against RNA molecules detected. Each regression line was calculated from a triplicate data set. Depicted
experiments were performed by Dr. Manfred Weidmann and coworkers, University of Göttingen.
Comparison of the efficiencies of the Dobrava assays (E =10(-1/slope)-1) of 0.56 and 0.61
for a Tth based kit and TaqM1 in bicine buffer, however clearly indicate that the novel
TaqM1 enzyme shows a real-time RT-PCR performance comparative to the aptamer
blocked Tth DNA polymerase.
55
Results and discussion
Due to the high transcription temperatures possible with this novel enzyme, Taq M1
has a high potential to develop into a very good real-time RT-PCR enzyme, especially
for the detection of secondary structure prone RNA molecules of RNA viruses or tmRNA in bacteria146,147.
2.4.3
Conclusion
A wild-type 5´-3´-nuclease domain was successfully combined with a previously
described N-terminal shortened mutated Taq DNA polymerase28 that has significantly
increased reverse transcriptase activity without significantly compromising
polymerase and nuclease function of the resulting chimera Taq M1. It was
demonstrated that Taq M1 has the similar PCR activity as the Taq wt enzyme.
Furthermore, the mutations in the polymerase domain have little effect on the activity
of the attached 5´-3´nuclease domain. It was demonstrated that Taq M1 can be used
for reverse transcription of RNA targets at high temperatures (~60-70°C). The
5´-3´nuclease domain of Taq M1 renders this enzyme highly suitable for any probe
based detection methods. This was demonstrated in the detection of RNA pathogens
from natural sources. Noteworthy, without further optimisation of parameters,
comparable detection sensitivities than commercially available one-step RT-PCR
systems, which are usually based on enzyme blends, were found. The system might be
further enhanced by optimizing the reaction buffer composition, reaction conditions
like pH and reagent concentrations. Further advancements of RNA detection by onestep RT-PCR in particular of complex RNA targets with highly stable secondary
structure motifs in which reverse transcription at high temperatures is of urgent need.
The scaffold of Taq M1 could serve as the basis for further progress along these lines
employing directed enzyme evolution31,39,46-48,148.
A manuscript for publication of these results has been submitted.
56
Results and discussion
2.5
Functional Studies on the Tyrosin Y671 responsible for A-rule in
KlenTaq DNA polymerase
2.5.1
Introduction
Replicative DNA polymerases are usually synthesising DNA in a template dependent
manner according to Watson and Crick base pairing. Two exceptions are described for
the unusual case of non-templated DNA synthesis by replicative DNA polymerases:
Abasic site bypass and overhang DNA synthesis.
Abasic sites are the most frequent DNA lesions formed in the genome149 and are the
result of hydrolysis of the glycosidic bond that connects the nucleobase with the sugar
moiety. It has been estimated that approximately 10000 abasic sites per day are
formed in a human cell150. Most are sensed and excised by DNA repair systems using
the sister strand to guide incorporation of the right nucleotide in the position of the
lesion, but sometimes abasic sites stay undetected and cause problems in DNA
replication. Abasic sites are strong barriers for replicative DNA polymerases. Native
abasic sites are instable and may lead to strand breaks. That is why in most research
studies the stabilized tetrahydrofurane analogue (Figure 38) is used. It has been
shown in several studies that adenine is most frequently incorporated opposite of that
lesion followed by guanine, a behaviour which is termed as ’A-rule’ 4,132. However, the
mechanism that is responsible for purine selection by DNA polymerases is not fully
understood.
Figure 38
Hydrolysis of the glycosidic bond leads to loss of the coding nucleobase and
formation of an abasic site (left). Stabilised abasic site analogue (right).
Gloeckner et al.151 recently described an N-terminal shortened mutated Taq DNA
polymerase (KlenTaq DM KK) from Thermus aquaticus which showed significantly
increased ability to bypass DNA lesions such as the described abasic site. The mutant
harbours two mutations in amino acid residues binding to the DNA substrate (I614K
57
Results and discussion
and M747K). In both cases hydrophobic amino acids (isoleucine and methionine,
respectively) having van der Waals contacts with the backbone of the nucleotide or
DNA substrate were substituted by a polar, positively charged lysine. KlenTaq DM KK
was mainly characterized by radiometric primer extension analyses. It was found that
KlenTaq DM is > 50 times more efficient in incorporating dATP nucleotides opposite the
abasic site than the wild-type enzyme151.
Furthermore, it is known that 3’-5’-exonuclease deficient DNA polymerases usually add
an additional nucleotide to a blunt-ended primer template complex in a nontemplated manner121 preferentially incorporating dATP, thus producing a single
stranded overhang at the 3´end (see Figure 39).
Figure 39
Blunt-ended primer template DNA complex. 3´-5´exonuclease deficient DNA
polymerases add an additional nucleotide in a non-templated manner, preferentially dA121.
Intrinsic properties of adenine like superior stacking ability of the incoming adenosine
nucleotide to the nucleobase system at the primer end as well as solvation were
proposed as being the driving forces for preferential adenine selection. Until now, in
reactions with blunt-ended or the abasic site substrate all known explanations for the
preferential purine selection are still unconvincing.
Recently, Schnur et al.152 crystallised KlenTaq DM incorporating either an adenine or
guanine opposite an abasic site substrate. Additionally, Obeid et al.153 was recently
able to crystallise KlenTaq DM bound to a similar DNA duplex structure as depicted in
Figure 39. In both situations it was found that a tyrosine (Y671) fills the space of the
absent template nucleobase both at the abasic site and at the blunt-ended DNA
substrate of the nascent nucleobase pair, thereby mimicking a pyrimidine nucleobase
in shape and size.
2.5.2
Results
Inspired by the crystal structures obtained from Schnur and Obeid, in which a tyrosine
Y671 fills the space of the absent template, KlenTaq wild-type and KlenTaq DM were
58
Results and discussion
first tested in radiometric primer extension reactions for incorporation of the four
different triphosphates (dATP, TTP, dCTP and dGTP, see Figure 40).
Figure 40
(A) Partial primer template sequence used in the dNTP incorporation opposite
abasic site experiment. (B) Single nucleotide incorporation of KlenTaq wild-type and DM opposite abasic
site at identical conditions at 72°C for 0.5, 2, or 10 min., respectively. The respective enzyme (50 nM) and
dNTP (100 μM) are indicated.
Both enzymes were incubated separately with respective dNTPs at 72°C for increasing
time periods (0.5, 2 and 10min). Indeed, the fastest incorporation opposite an abasic
site analogue is found for dATP. Thus, wild-type and DM followed the A-rule and
preferentially incorporated dA in a non-templated manner as expected. Furthermore,
DM incorporated dATP much faster than the wild-type enzyme producing the 24 nt long
product in only 30sec., which is in line with previous kinetic measurements151 that
KlenTaq DM is > 50 times more efficient in incorporating dATP nucleotides opposite the
abasic site than the wild-type enzyme.
Next, wild-type and DM was studied in reactions with a blunt-ended DNA substrate to
study 3´ overhang synthesis (see Figure 39 and Figure 41). Again, both enzymes were
incubated separately with respective dNTPs at 72°C for increasing time periods (0.5, 4,
15 and 60min; see Figure 41). As in the case of an abasic site substrate, both enzymes
preferred incorporation of dA at the blunt-ended DNA substrate. KlenTaq wild-type
produces mainly a 24nt long product in presence of dATP after 1h incubation time,
whereas DM generates the same product after 30sec. of incubation. Interestingly,
during incubation of KlenTaq DM with dCTP a faint band of a 25 nt long product is also
formed.
59
Results and discussion
Figure 41
(A) Partial primer template sequence used in the dNTP incorporation assay at
blunt-ended DNA substrate. (B) Single nucleotide incorporation of KlenTaq wild-type and DM at the bluntended DNA substrate at identical conditions at 72°C for 0.5, 4, 15 and 60 min., respectively. The respective
enzyme (100 nM) and dNTP (200 μM) are indicated.
Due to the finding that both at the abasic site and at the blunt-ended DNA substrate a
tyrosine (Y671) fills the space of the absent template nucleobase of the nascent
nucleobase pair (Schnur and Obeid152,153), amino acid sequence alignment was
performed (see Figure 42). It was found that in all A-family DNA polymerases from
prokaryotic and eukaryotic origins the corresponding O helices are highly conserved
throughout evolution from bacteria to humans154. Especially the respective aromatic
residue Y671 is evolutionary highly conserved.
Figure 42
Amino acid sequence alignment of DNA polymerases covering position Y671
from Thermus aquaticus Polymerase I.
To point out that Y671 plays the most important role for the A-rule behaviour in
KlenTaq DNA Polymerase, two mutants of KlenTaq were constructed by site directed
mutagenesis: The Y671A mutant was constructed in order to test the requirement of an
aromatic residue at this position. The second mutant Y671W was constructed to
explore a possible molecular mimicry mechanism in a way that the tyrosyl-side chain
60
Results and discussion
Y671 of the DNA polymerase directs for incorporation of a purine opposite the missing
template base (see Figure 43).
Figure 43
Chemical structures of purines (A,G) and pyrimidines (C,T) compared with
tyrosine (Y) and tryptophane (W) demonstrating similar steric demands.
The mutation Y671W will transform the six-membered phenol ring in tyrosine into a
bicyclic indole in tryptophane, consisting of a six-membered ring fused to a fivemembered ring. If a mimicry mechanism is at work this would result in an approximate
size of adenine or guanine and should thus mimic a purine residue instead of a
pyrimidine nucleobase:
First, the resulting mutants were over-expressed in E. coli cells, purified by Ni-NTA
affinity chromatography and visualised on SDS-PAGE to control for purity and
concentration adjustment by comparing to a BSA standard dilution series (see Figure
44).
Figure 44
SDS-PAGE gel of purified KlenTaq DNA polymerases wild-type (wt), Y671A and Y671W.
Again, both mutants Y671A and Y671W were tested in radiometric primer extension
reactions for incorporation of the four different triphosphates (dATP, TTP, dCTP and
dGTP).
61
Results and discussion
Figure 45
(A) Partial primer template sequence used in the dNTP incorporation opposite
abasic site experiment. (B) Single nucleotide incorporation of KlenTaq Y671A and Y671W opposite abasic
site at identical conditions at 72°C for 2, 10 and 60 min., respectively. The respective enzyme (500 nM)
and dNTP (100 μM)are indicated.
Indeed, mutation Y671A led to an enzyme that has significantly reduced activity in
nucleotide incorporation both at the blunt-ended duplex and with the abasic site
substrate (see Figure 45 and Figure 46, left side). Only very less product formation was
observed after incubation with dGTP or dATP at 72°C for 60min and longer in both
cases, whereas in the case of Y671W the preference for dA vanished and instead the
pyrimidines TTP and dCTP are most efficiently incorporated (see Figure 45 and Figure
46, right side). Interestingly, during incubation of mutant Y671W with dCTP and TTP, a
faint band indicating a 25 nt long product is also formed, which is similar to the
findings with KlenTaq DM above.
62
Results and discussion
Figure 46
(A) Partial primer template sequence used in the dNTP incorporation assay at
blunt-ended DNA substrate. (B) Single nucleotide incorporation of KlenTaq Y671A and Y671W at the bluntended DNA substrate at identical conditions at 72°C for 4, 15,60 and 240 min., respectively. The respective
enzyme (100 nM) and dNTP(200 μM) are indicated.
Kinetic studies with KlenTaq wild-type and Y671W with blunt-ended DNA duplex
substrate (see Figure 46, (A), above) were performed to further evaluate the changed
substrate preferences of the mutant Y671W from an A-rule to a T-rule behaviour (see
Table 5).
Table 5
Nucleotide incorporation at the blunt-ended DNA duplex substrate
enzyme
dNTP
kpol
Kd
kpol/Kd
[s-1·10-2]
[μM]
[s-1·μM-1·10-4]
wt
dATP
11.4 ± 0.86
240 ± 33.2
4.75
wt
TTP
0.72 ± 0.15
838 ± 258
0.09
Y671W
dATP
0.49 ± 0.06
630 ± 122
0.08
Y671W
TTP
1.63 ± 0.31
934 ± 251
0.17
While dT is incorporated at the blunt-ended substrate by the wild-type enzyme with
~50-fold lower efficiency (kpol/KD) compared to dA, the opposite is observed for Y671W.
The mutant enzyme shows a 3-fold higher efficiency for dT incorporation compared
with dA.
2.5.3
Conclusion
These findings corroborate the model of a nucleobase mimicry mechanism for the
phenomenon of a non-templated DNA synthesis from a template-dependent replicative
DNA polymerase. These results further highlight the importance of a geometric fit of
63
Results and discussion
the substrates to the active site of the enzyme for DNA polymerase activity. In accord
with these observations, it has been shown that DNA polymerases are able to process
non-natural nucleobase surrogates placed in template strands that mimic the shape
and size of the natural nucleobase but have decreased hydrogen bonding
capabilities155. Due to the nucleobase mimicry of Y671, purines more favourably fill the
vacant space in the binding pocket than the smaller pyrimidines thymine and cytosine.
The preference for adenine over guanine incorporation opposite abasic sites (A-rule)
can be rationalized by the superior stacking and solvation properties of adenine over
guanine, as envisioned before155. Furthermore, the aromatic residue Y671 is
evolutionary highly conserved. Amino acid sequence alignment of A-family DNA
polymerases from prokaryotic and eukaryotic origins showed that the corresponding O
helices and especially Y671 is highly conserved throughout evolution from bacteria to
human. Due to the high conservation in DNA polymerases from prokaryotes,
eukaryotes and archaea, it is likely that the depicted mechanisms of non-templated
DNA synthesis are general and apply to other DNA polymerases in this sequence family
as well. Especially in the case of abasic sites, the findings are of physiological
relevance. The depicted mimicry of nucleobase shape and size has not been reported
previously for a translesion synthesis. This mechanism to use protein residues to direct
nucleotide incorporation is reminiscent to a transfer RNA CCA-adding enzyme156 and
the specialized DNA polymerase Rev1 that exclusively incorporates cytosine
nucleotides157. Other DNA polymerases such as of the Y-family are known to bypass
several DNA lesions. However, they do not follow the A-rule in abasic site bypass4,132
and in contrast use the nucleotide 5’ to the abasic site as template. Thus, their
selectivity cannot account for the A-rule observed in studies described above.
In summary, the purine specificity of non templated DNA synthesis, especially for the
physiological very important abasic site translesion synthesis, in DNA polymerases
from the A-family stems from specific interactions of the incoming dNTP with the
protein side chain that mimics the size and shape of the absent nucleobase rather than
the DNA.
A manuscript for publication of these results is currently under preparation.
64
Summary and outlook
3
Summary and outlook
In this PhD thesis, several projects about the functional analysis and recruitment of
mutated DNA polymerases for improved biotechnological applications were
investigated.
Discrimination of incorrect pairing single nucleotides is of fundamental importance for
the enzyme-aided detection of single nucleotide variations (single nucleotide
polymorphisms (SNPs)). It could be demonstrated that both chemically modified
primer probes which are thiolated at the 2-position of thymidine as well as mutated
DNA polymerases were able to increase single-nucleotide discrimination22.
Based on these findings a DNA chip based system for the multiplex detection of single
nucleotide polymorphisms (SNPs) was established77. For that purpose, a mutated DNA
polymerase from Pyrococcus furiosus with improved single nucleotide discrimination
properties72 is used for selective microarrayed primer extensions. It is shown that the
mutated DNA polymerase in combination with unmodified primer strands fulfils the
demands on solid support and obviates the need for chemical modifications of the
primer probes as required before22-24,74,76,104. The system depicted herein could provide
the basis for further advancements in microarrayed nucleic acid diagnostics using
tailor-made enzymes.
Until now, all reported methods for DNA polymerase evolution are restricted to a single
enzyme property, for example, increased selectivity or the ability to efficiently process
DNA lesions28,31,39-44,46,50. Thus, a new microarrayed device was developed to overcome
these obvious limitations that allow the multiplexed screening of several enzyme
features in parallel:
The approach is based on the spatial separation of different covalently attached DNA
substrates on a glass slide and their selective addressing by oligonucleotide
hybridization. This system, termed oligonucleotide-addressing enzyme assay (OAEA),
enables multiplexed simultaneous profiling of DNA polymerases in nanoliter volumes
in terms of their different properties. OAEA can be used for the simultaneous and
multiplexed profiling of several enzyme features with high throughput. Additionally,
65
Summary and outlook
other DNA-modifying enzymes like ligases and endonucleases can be included in
multiplex directed evolution approaches using OAEA. As a first successful
demonstration it was used to identify enzymes with altered properties out of a library
of DNA polymerase mutants148.
A functional chimeric DNA polymerase could be obtained by fusion of a wild-type 5´3´nuclease domain with a recently described N-terminally shortened DNA
polymerase28 from Thermus Aquaticus, which exhibits a significantly increased reverse
transcription activity. The new enzyme (named as Taq M1) was created to improve RNA
pathogen detection systems for pathogens like Dobrava viruses. It could be
demonstrated that the fusion of polymerase- and 3´nuclease-domain to constitute Taq
M1 has no effect on the originally polymerase- and nuclease function and activities.
Additionally, Taq M1 was used in applied TaqMan RNA detection assays: Without
optimisation of reaction conditions Taq M1 provided detection sensitivities compared
to commercially available one-step RT PCR systems, which are based on enzyme blends.
Taq M1 is highly recommended for the use of one-step RT PCR, especially if high
transcription temperatures are desired to melt stable secondary structures of RNA
targets.
In my last project, functional studies were conducted with the N-terminally shortened
DNA polymerase from Thermus Aquaticus (KlenTaq). Recently obtained crystal
structures of KlenTaq in complex with both an abasic site harbouring template and a
blunt-ended primer template substrate (Schnur et al.152 and personal communication
with S.Obeid153), revealed that amino acid tyrosine 671 plays an important role in the
template-less selection of the incorporated nucleotide. Tyrosine 671 thereby mimics
the steric constraints of a pyrimidine template base resulting in the favoured
incorporation of purine bases (A and G). Mutation of tyrosine into alanine (Y671A)
results in a dramatic drop of catalytic activity. Mutation of the aromatic tyrosine into
the also aromatic but steric more demanding tryptophane results in the favoured
incorporation of pyrimidine bases (T and C). These findings could be proved by single
nucleotide incorporation studies and enzyme kinetic measurements.
66
Summary and outlook
4
Zusammenfassung und Ausblick
In dieser Doktorarbeit wurden verschiedenste Projekte bearbeitet, die die funktionale
Analyse und den Einsatz von mutierten DNA-Polymerasen in biotechnologischen,
diagnostischen Anwendungen untersuchen:
Für die enzymatische Detektion von Einzelbasen-Varationen (SNPs, engl.: single
nucleotide polymorphisms) ist die Diskriminierung von fehlgepaarten Einzelbasen von
fundamentaler Bedeutung. Es konnte gezeigt werden, dass die Diskriminierung der
fehlgepaarten Einzelbasen sowohl durch chemisch modifizierte Primersonden, die
beispielsweise thiolierte Thymidine tragen22, als auch genetisch durch mutierte DNAPolymerasen erhöht werden kann. Darauf aufbauend wurde unter anderem ein auf
DNA-Chips basierendes Verfahren etabliert, welches die vielfache, synchrone Detektion
von SNPs ermöglicht77. Hierbei konnte eine mutierte DNA-Polymerase aus dem
Organismus Pyrococcus furiosus mit einer erhöhten Diskriminierungsfähigkeit
gegenüber fehlgepaarten Einzelbasen72 erfolgreich eingesetzt werden. Das System aus
mutierter DNA-Polymerase und unmodifizierten Primersträngen genügt der
verlässlichen Detektion und ersetzt somit die Notwendigkeit des Einsatzes von
chemisch modifizierten Primersträngen22-24,74,76,104. Dieses Verfahren legt einen
wegweisenden Grundstein für den Einsatz von maßgeschneiderten Enzymen in der
modernen Nucleinsäure-Diagnostik.
Bisher sind alle bisher bekannten Methoden der gerichteten Evolution von DNAPolymerasen auf eine einzige Enzymeigenschaft beschränkt, wie zum Beispiel der
erhöhten Diskriminierungsfähigkeit gegenüber fehlgepaarten Einzelbasen28,31,3944,46,50
. Um diese deutliche Einschränkung zu umgehen, wurde ein Mikroarray-basiertes
Verfahren
entwickelt,
das
das
vielfache
Screenen
von
verschiedensten
Enzymeigenschaften zeitgleich und parallel ermöglicht. Dieses Verfahren ermöglicht
die selektive Hybridisierung von Oligonukleotiden durch kovalent befestigte und
räumlich getrennte DNA Substrate und wird als „Oligonukleotid adressierender Enzym
Assay“ (OAEA, engl. oligonucleotide-addressing enzyme assay) bezeichnet. Mit OAEA
67
Summary and outlook
können simultan verschiedenste DNA-Polymerase Eigenschaften in kleinsten Volumina
(Nanoliter Mengen) im Hochdurchsatz gescreent werden. Zusätzlich besteht die
Möglichkeit der Übertragung des Systems auf andere DNA-prozessierenden Enzyme wie
Ligasen oder Nucleasen. Um die Praktikabilität zu demonstrieren, wurden in einer
ersten
Testreihe
DNA-Polymerase-Mutanten
mit
gänzlich
gegensätzlichen
Eigenschaften, die aus einer kleinen Enzymbibliothek stammen, erfolgreich
identifiziert und charakterisiert148.
Durch die Fusion einer Wildtyp 5´-3´Nuclease Domäne mit einer zuvor beschriebenen
N-terminal verkürzten DNA Polymerase Mutante28 von Thermus Aquaticus, die eine
signifikant erhöhte reverse Transkriptase Aktivität besitzt, konnte eine funktionale
chimäre DNA Polymerase erhalten werden. Das neue Enzym (genannt Taq M1) wurde
erzeugt, um eine verbesserte RNA-Pathogen Detektion, zum Beispiel von Dobrava- und
Gelbfieber-Viren, zu ermöglichen. Es konnte gezeigt werden, dass die Fusion zur Taq
M1 ohne Verlust von ursprünglicher
Polymerase- oder
Nuklease-Funktion
bewerkstelligt wurde. Zudem wurde Taq M1 in angewandten TaqMan RNA
Detektionsassays getestet: Ohne Optimierung an Reaktionsbedinungen lieferte Taq M1
vergleichbare Detektionsergebnisse wie kommerziell erhältliche one-step RT-PCR
Systeme, welche auf den Einsatz von Enzymmischungen beruhen. Durch ihre
Thermostablität empfiehlt sich Taq M1 somit für den Einsatz von one-step RT-PCR
insbesondere dann, wenn erhöhte Transkriptionstemperaturen gewünscht sind, um
besonders stabile Sekundärstrukturen des RNA Targets aufzuschmelzen.
In meinem letzten Projekt wurden funktionale Studien an der N-terminal verkürzten
DNA-Polymerase von Thermus aquaticus (KlenTaq) durchgeführt. Durch zwei kürzlich
erhaltene Kristallstrukturen der KlenTaq Polymerase sowohl mit einer abasischen
Stelle- als auch einem blunt-end enthaltenen DNA-Templat konnte gezeigt werden,
dass die Aminosäure Tyrosin671 eine wichtige Rolle in der templatlosen Auswahl des
inkorporierten Triphosphates spielt (Schnur et al.152 und S. Obeid153 persönliche
Kommunikation). Das Tyrosin imitiert den sterischen Anspruch einer auf der
Templatstrangseite stehenden Pyrimidinbase, was im bevorzugten Einbau von
Purinbasen (A und G) resultiert. Durch die Mutation des Tyrosins zu Alanin verliert das
68
Summary and outlook
Enzym stark an katalytischer Aktivität. Eine Mutation des Tyrosin zu dem sterisch
anspruchsvolleren Tryptophan hingegen resultiert zu dem bevorzugten Einbau von
nunmehr Pyrimidinbasen (T und C). Dies konnte durch funktionale Einzeleinbaustudien
demonstriert und durch Kinetikmessungen untermauert werden.
69
Materials and methods
5
Materials and methods
5.1
Reagents
All chemicals were - if not stated otherwise - of p.a. or of molecular biology quality
grade. Purified water was drawn from a combined reverse osmosis/ultrapure water
system (Sartorius stedim biotech,arium-series).
Reagent
Supplier
Acrylamide
Roth
Acetic acid
Prolabo Normapur
Agar
Roth
Agarose
Invitrogen
Ammonium hydroxide solution (33%)
Riedel de Haën
Ammonium peroxodisulfate
Fluka
Anhydrotetracycline
IBA
β-Mercaptoethanol
Roth
Boric acid
Fluka
Bromphenol Blue
Fluka
Bovine Serum Albumin Standard (2 mg/ml)
Pierce
Carbenicilline disodium salt
Roth
Chelating Sepharose Fast Flow
GE Healthcare
Coomassie Brillant Blue G 250
Roth
1,4-Dithiothreitol
Roth
5’-O-(4,4’-Dimethoxytrityl)-(3-N/4-O-toluoyl)-
Berry & Associates (USA)
2-S-thiothymidine
5’-O-(4,4’-dimethoxytrityl)-4-S-(2-cyanoethyl)
Berry & Associates (USA)
thiothymidine
Dimethylformamide
Merck
Dimethylsulfoxide
Fluka
Ethanol (100%)
Roth
70
Materials and methods
Reagent
Supplier
Ethidiumbromide (0.1%)
Roth
Ethylenediaminetetraacetic acid (EDTA)
Roth
Formamide
Merck
Glycerol
Prolabo
Glycine
Roth
Imidazole
Merck
Isopropyl β-D-1-thiogalactopyranoside (IPTG)
Roth
LB broth (Lennox)
Roth
Magnesium chloride
Acros Organics
Ni-NTA-Agarose (see Chelating Sepharose)
N,N,N‘,N‘-Tetramethylenethylendiamine
Roth
1-Propanol
Sigma
reagents for solid phase oligonucleotide Applied Biosystems, Vivotide or
synthesis
JT Baker.
Sodium acetate trihydrate
Merck
Sodium dodecyl sulfate (SDS)
Roth
Sodium hydroxide
Merck
Sephacryl S-300 High Resolution
Amersham
Sephadex-G-25 Superfine
GE Healthcare
SYBRgreenI
Molecular Pobes
Self seal reagent
MJ Research
Tris(hydroxymethyl)aminomethane (TRIS)
Roth
Triton X-100
Roth
Tryptone
Roth
Tween 20
Riedel de Haën
Urea
Roth
Xylencyanol
Roth
Yeast extract
Roth
71
Materials and methods
Nucleotides and radiochemicals
Supplier
dNTPs and NTPs
Roche and Fermentas
[γ-32P]-ATP
Hartmann Analytic
5.2 Biochemical reagents, enzymes and kits
Kit
Supplier
Antarctic Phosphatase
New England Biolabs
BsmBI
New England Biolabs
DpnI
NEB / Fermentas
EcoRV
New England Biolabs
Gene Ruler DNA Ladder Mix
Fermentas
Gene Ruler 1 kp DNA-Leiter
Fermentas
High Pure PCR Cleanup Micro Kit
Roche
High Pure Plasmid Isolation Kit
Roche
Lysozyme
Sigma
PageRuler Unstained Protein Ladder
Fermentas
Phusion DNA Polymerase
Finnzyme
QIAquick Gel Extraction Kit
Qiagen
RNA from Bacteriophage MS2
Roche
Rapid DNA Ligation Kit
Fermentas
T4 Polynukleotidkinase
New England Biolabs
Vent (exo-) DNA polymerase
New England Biolabs
5.3
Bacterial strains and plasmids
Bacterial strain and plasmids
Source
electro competent E. coli XL10-Gold
Stratagene
(kindly
provided
by
Staiger)
E. coli BL21 (DE3) Gold
Stratagene
pGDR11
AG Welte, University of Konstanz158
pASK-IBA37plus
IBA
72
N.
Materials and methods
5.4
Disposables
Disposable
Supplier
96-well plates (200 μl)
ABgene
96-deep well plates (2.2 ml)
Peske
384-deep well plates (300 μl)
Abgene
Adhesive sealing foil
ABgene
Cuvettes (1.5 – 3 ml)
Roth
Diamond sealing foil
ABgene
Electroporation Gene Pulser cuvettes (1/2 mm)
BioRAD
Gas permeable adhesive seals
ABgene
Glass wool
Serva
Injection needles
Braun
Petri dishes
Roth
Reaction tubes (1.5, 2.0 ml)
Peske Laborbedarf
Scalpels
Bayha
Sephadex G-25 columns
GE Healthcare
Tips for pipetting robot
Hamilton Robotics
Tips for multichannel pipettes
Peske
Tips for laboratory pipettes
Peske, Eppendorf
UV-cuvettes
Eppendorf
Vivaspin columns (6, 20 ml) 10.000 MWCO
Sartorius stedim biotech
Whatmanpaper
3mm Merck Eurolab
5.5
Equipment
Instrument
Supplier
Agarose gel racks
Fisher Scientific, ABgene
Autoclav
Autoclav Systec 3150 ELV
Chircular Dichroism Spectrometer, Jasco 720
Jasco Inc., Easton Maryland
Electroporator Gene Pulser Xcell
BioRAD
73
Materials and methods
Instrument
Supplier
ESI-MS 3000
Bruker Daltonics
Floor centrifuge 5804R
Eppendorf;
Freezer (-80°C, -20°C, 4°C)
Thermo Forma, Liebherr, Premium
Gel documentation device, Chemidoc XRS
BioRAD
Gel dryer
BioRAD
GenePix Personal 4100A microarray scanner
Molecular Devices
Heating block
Stuart
Incubation shaker
New Brunswick Scientific
Incubation shaker, Titramax 1000
Heidolph
Laboratory pipettes
Eppendorf
Magnetic stirrer MR 3000 D
Heidolph
Microwave oven
Micromaxx
Microarray Peltier Thermal Cycler 200
MJ Research
Multifuge KR 4
Heraeus
Multichannel pipettes Transferpette
Brand
Nanoplotter 2.0 system
GeSiM
equipped with:
Thermostat Unistat Tango
Huber Kältemaschinenbau
Humidity Control II
Lucky Reptile
Mist generator Hygro Plus
Hobby
Oligonucleotide synthesizer
ABI 392
PAGE electrophoresis racks
BioRAD
PCR-thermocycler
Biometra
pH meter, Seven Easy
Mettler Toledo
Phosphorimager Molecular Imager Chemi-Doc BioRAD
XRS System
Photometer Cary 100 Bio
Varian
Pipetting robot Microlab Star
Hamilton Robotics
Phosphor screens
Fuji
74
Materials and methods
Instrument
Supplier
Phosphor screen cassettes BAS-Cassette 2025
Fuji
Plate sealer
Abgene
Power supply Power Pac 3000
BioRAD
Radioactivity counter Contamat FHT111M
Thermo
Reagent dispenser Multidrop
Thermo
Real-time PCR-thermocycler:
Chromo4
BioRAD
Lightcycler 480
Roche
Refrigerated centrifuge Biofuge Primo R
Heraeus
Radioactivity shields
Roth
SDS-PAGE racks
BioRAD
Speed-Vac Concentrator 5301
Eppendorf
Sterile bench
HERA safe
Thermomixer, Thermomixer comfort
Eppendorf
Table top centrifuges, MiniSpin
Eppendorf
UV-transilluminator
Bachofer
UV/VIS photometer ND-1000 Nanodrop
Peqlab
Vortexer REAX Control
Heidolph
Water baths
Memmert
75
Materials and methods
5.6
Buffers and solutions
Buffers and solutions
Components
Concentration
Agarose gel loading buffer
Bromophenol Blue
0.025% (w/v)
Xylene cyanol FF
0.025% (w/v)
Glycerol
60%
EDTA
60 mM
Agarose
gel
staining Ethidium bromide
0.5 μg/ml in 1x TAE
staining Acetic acid
50 ml
solution
Coomassie
solution
Coomassie
125 ml
Water
300 ml
Coomassie Roti Blue
375 mg
destaining Acetic acid
solution
10x KlenTaq
buffer
Ethanol
Ethanol
reaction Tris-HCl pH 9.2
1 x KlenTaq lysis buffer
10%
30% (v/v)
500 mM
(NH4)2SO2
160 mM
MgCl2
25 mM
Tween 20
1 % (v/v)
1 x KlenTaq reaction buffer
Lysozyme
2 x KLenTaq washing Tris-HCl pH 9.2
buffer (Ni-NTA purification
MgCl2
and Vivaspin)
Imidazole
0.1 mg/ml
100 mM
25 mM
various concentrations: (0,
5, 20,200 mM)
1x KlenTaq storage buffer
76
Tris-HCl pH 9.2
50 mM
(NH4)2SO2
16 mM
MgCl2
2.5 mM
Tween 20
0.1 % (v/v)
Glycerole
~ 50% (v/v)
Materials and methods
SDS-PAGE
stacking
gel Tris-HCl pH 6.8
1M
buffer
SDS-PAGE resolving gel Tris-HCl pH 8.8
1.5 M
buffer
SOB-Medium pH 7.0
Tryptone
2% (w/v)
Yeast extract
0.5% (w/v)
NaCl
0.05%(w/v)
After sterilisation,
addition of:
SOC-Medium pH 7.0
MgCl2
0.01M
MgSO4
0.01M
SOB Medium +
Glucose 20%(w/v)
5x
SDS-PAGE
loading Tris-HCl pH 8.0
buffer
10x
10x TBE buffer
10x
10x TBE buffer
0.5 M
Glycerol
50%
Bromophenol Blue
0.05% (w/v)
ß-Mercaptoethanol
1% (v/v)
250 mM
Glycin
2M
SDS
1% (w/v)
Tris
890 mM
Boric acid
890 mM
EDTA pH 8.0
20 mM
SDS-PAGE Tris-HCl pH 8.9
electrophoresis buffer
1M
EDTA
SDS-PAGE Tris-HCl pH 8.9
electrophoresis buffer
2% (v/v)
250 mM
Glycin
2M
SDS
1% (w/v)
Tris
890 mM
Boric acid
890 mM
EDTA pH 8.0
20 mM
77
Materials and methods
Buffers and solutions
Components
Concentration
Urea-PAGE loading buffer EDTA
20 mM
(STOP-solution)
Formamide
80%
Bromophenol Blue
0.025% (w/v)
Xylene cyanol FF
0.025% (w/v)
Urea-PAGE stock solution 1 Urea
8.3 M in 10x TBE
Urea-PAGE stock solution 2 Acrylamide solution
25%
Urea
8.3 M
N,N´-
2%
Methylenbisacrylamide
1x Pfu reaction buffer
Tris-HCl pH 8.8
20 mM
MgSO4
2 mM
(NH4)2SO4
10 mM
KCl
10 mM
Triton-X100
0.1% (v/v)
BSA
0.01 mg/ml
Urea-PAGE stock solution 3 Urea
5.7
8.3 M
Determination of DNA concentration
Quantification of DNA and RNA was conducted by UV absorption measurements at 260
nm using UV/VIS photometer. Water or the respective buffer was used as a reference.
Based on Lambert-Beer's law the DNA concentration could be calculated. Extinction
coefficients, as well as molecular weight and melting behaviour were determined using
the
online
available
“Oligonucleotide
Properties
Calculator”
(http://www.basic.northwestern.edu/biotools/oligocalc.html)159 which is based on
specific extinction coefficients of each different nucleobase.
78
Materials and methods
5.8
Oligonucleotides
Oligonucleotides were purchased from Metabion international AG (Martinsried,
Germany) or Purimex (Grebenstein, Germany). All chemically modified oligos, oligos
for allele specific reactions and kinetic characterisations were purified by denaturing
PAGE. Templates and cloning primers were used desalted or 1x HPLC purified.
The integrity of all oligonucleotides was confirmed by ESI-MS. Therefore,
oligonucleotide samples were diluted (c = 1μM) in a mixture of water (79% v/v), 2propanol (20% v/v) and triethylamine (1% v/v).
5.9
Radioactive labelling of DNA-oligonucleotides
Oligonucleotides were radioactively labelled by [32P]-ATP using T4 polynucleotide
kinase (T4-PNK). The labelling reaction was carried out in 50 μl scale with 400 nM
oligonucleotides, 20 μCi [32P]-ATP (2 μl), 0.4 U / μl T4-PNK (2 μl) and 1x T4-PNK
reaction buffer. The reaction was incubated at 37°C for ~1 h and subsequently stopped
by heat-inactivation of T4-PNK at 95°C for ~5 min. After gel filtration (G25 sephadex
spin column) to remove salt and residual [32P]-ATP, the radioactively labelled
oligonucleotide solution was diluted with 19 μl of a 10 μM unlabelled oligonucleotide
. applied RT temin a final primer concentration of 3 μM.
5.10
Agarose gelelectrophoresis
Agarose gel electrophoresis was performed according to standard procedures.
Depending on fragment DNA size a 0.8% or 2.5% agarose in 0.5 x TBE was used. Gels
were stained for 5-30 min in ~0.01% (w/v) ethidiumbromide (EtBr) in 0.5 x TBE and
shortly destained in 0.5 x TBE for further 5 min and documented in a Chemidoc XRS
System. For preparative agarose-gels it was taken care that UV irradiation of the
fragments to be isolated was minimised.
5.11
Denaturing polyacrylamide gelelectrophoresis
Denaturing polyacrylamide gel electrophoresis (PAGE) was performed according to
standard procedures. It was applied for purification of DNA–oligonucleotides and for
analytical separation of reaction products. All denaturing polyacrylamide gels (12%)
79
Materials and methods
contained urea. Preparative gels had a gauge of 1.5 mm and analytical gels a gauge of
0.4 mm.
After running, analytical gels were transferred onto whatman paper, dried
subsequently and exposed to a phosphor screen preferentially overnight.
For DNA-isolation from polyacrylamide-gels the separated DNA was visualised by UVshadowing and the respective bands were excised with a scalpel. Excised gel pieces
were crushed by forcing them through a syringe and collected in 2 ml eppendorf tubes.
DNA was eluted from crushed gel pieces by adding H2O and incubation at 55 °C
overnight. Next, the DNA/PAGE mixture was filtrated through silanised glass-fibres
wool. The filtrate was dried in a speedvac (Eppendorf) and extracted DNA was purified
by ethanol precipitation according to standard procedures.
5.12
SDS polyacrylamide gelelectrophoresis
Expressed proteins were analysed by discontinuous glycine SDS-PAGE (12%) according
to standard procedures including a stacking- and a resolving-gel. Staining was
performed by a Coomassie-staining solution.
5.13
Determination of protein concentration
Protein samples were loaded in parallel with a BSA standard dilution series. Respective
band intensities were quantified on a BioRad Chemidoc XRS System (Quantity One
4.5.0) and protein concentrations could be calculated in comparison with the BSA
sample intensities by linear curve analysis.
5.14
Site directed mutagenesis
In general site directed mutagenesis was performed according to the QuikChange SiteDirected Mutagenesis protocol (Stratagene) with the following modifications: Desalted
mutagenesis primers and Phusion DNA Polymerase was used for whole plasmid PCR.
Negative controls were performed via plating DpnI digested original plasmid on LBagar plates with 100 ng/μl carbencillin.
80
Materials and methods
5.15
Transformation of chemically competent cells
Chemically competent E. coli cells were transformed as described in the manufacturer’s
manual (Stratagene) using an electroporator Gene Pulser Xcell.
5.16
DNA-sequencing
The prepared DNA samples were sent to a professional sequencing company (GATC
Biotech AG, Konstanz). Sequences were analysed and aligned using Chromas Lite
(http://www.technelysium.com.au/chromas_lite.html) and SDSC Biology Workbench
(http://workbench.sdsc.edu/).
5.17
Crystal structure models
Own DNA and protein crystal structure models were prepared by the respective PDBcode using PyMOL Molecular Viewer ( http://pymol.sourceforge.net/ ) for
visualisation.
5.18
Methods for section 2.1
5.18.1 Real-time PCR experiments
Real-time PCR was performed as described by using an iCycler system (BIORAD). In
brief, the reactions were performed in an overall volume of 20 μL containing 400 pmol
of the respective templates, in the appropriate buffer provided by the supplier for Vent
(exo-) DNA polymerase (20 mM tris(hydroxymethyl)aminomethane-HCl (Tris-HCl; pH
8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1%Triton-X100). The final mixtures
contained dNTPs (200 μM each of dATP, dGTP, dCTP and TTP), primers (0.5 μM each of
the respective primer probe and reverse primer), 0.4 units Vent (exo-) DNA polymerase
(New England Biolabs; units defined by the supplier) and a 1/50 000 aqueous dilution
of a SybrGreen I 10,000 solution in dimethylsulfoxide (DMSO). All PCR amplifications
were performed by employing the following program: Initial denaturation at 95°C for 3
min, followed by 40 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C
81
Materials and methods
for 35 s and extension at 72°C for 40 s. The presented results are from at least three
repeated independent measurements of duplicates that originated from one master
mix. Identical template-target, primer-probe and reverse-primer DNA sequences were
used to those employed in earlier studies for comparison24.
5.18.2 Primers and templates
Oligonucleotide Sequences are in the Farber disease109 context.
primer probes (20nt)
5’-d(CGTTGGTCCTGAAGGAGGAT*)with T*: T, 2ST, 4ST, TOMe, 2STOMe or 4STOMe.
reverse primer (20nt)
5’-d(CGCGCAGCACGCGCCGCCGT)
target template (90nt)
5’-d(CCGTCAGCTGTGCCG TCGCGCAGCACGCGCCGCCG TGGACAGAGGACTGCAGAAAATC AACCT
N TCCTCCTTCA GGACCAACGTACAGAG) with N= A or G.
template (33nt)
5’-d(AAATCAACCT N TCCTCCTTCAGGACCAACGTAC)-3’ with N= A or G.
5.18.3 DNA thermal-denaturation studies
Melting curves were recorded on a Cary 100 bio UV/Vis instrument with temperature
controller. Data were obtained from three individual cooling/heating cycles. Melting
temperatures (Tm values in °C) were obtained from the maximum of the first derivative
of the melting curves (absorbance at 260 nm versus temperature). Measurements were
conducted in the appropriate Vent (exo-) DNA polymerase buffer (ThermoPol, New
England BioLabs) without Triton-X100 (20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM
(NH4)2SO4, 2 mM MgSO4) and contained 4.75 μM duplex DNA. The mixtures were heated
at 95°C for 5 min and slowly cooled down to room temperature prior to the melting
curve measurements. A measurement of the buffer was conducted separately and
subtracted from the spectra resulting from the sample. Shorter templates (33nt, A/G)
and respective primer probes containing T, 2ST, 4ST, TOMe, 2STOMe or 4STOMe as above were
used.
82
Materials and methods
5.18.4 Circular dichroism spectra
CD spectra were recorded on a Jasco 720 instrument in ThermoPol buffer (New England
Biolabs; 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1%
Triton-X100) at room temperature. The samples contained 4.75 μM duplex DNA. All
mixtures were heated to 95°C for 5 min and allowed to cool slowly to room
temperature prior to measurements. A spectrum of the buffer was measured separately
and subtracted from the spectra resulting from the samples. An average of 12 spectra
was recorded in each experiment. The sequences were as described in the previous
Section 5.18.3.
5.18.5 Kinetic single-nucleotide incorporation studies
Single-nucleotide incorporation reactions contained varying enzyme amounts (0.8–8
nM) and primer (150 nM)–template (300 nM) complex in ThermoPol buffer. Primer–
template complexes were annealed and the reaction was initiated by addition of
different concentrations of dATP in solution. After incubation for different times at
72°C, reactions were quenched by addition of two reaction volumes of gel-loading
buffer (80% formamide (v/v), 20% water (v/v), 20 mM ethylenediamine tetraacetate
(EDTA)) and the product mixtures were analysed by 12% denaturating PAGE.
Incorporation quantities were measured by quantifying the intensity of each band
produced by the DNA polymerase by using a Phosphorimager. From this quantification,
the amount of incorporated nucleotide was calculated. The intensity of the background
was subtracted from each band. The reaction conditions were adjusted for different
reactions to allow 20% or less of primer extension, thereby ensuring singlecompleted-hit conditions according to published procedures111. Steady-state KM and
kcat values were obtained by fitting with the Hanes–Woolf equation. The presented
results are from measurements that were repeated independently at least three times.
83
Materials and methods
5.19
Methods for section 2.2
5.19.1 Primers and templates
The integrity of all primer probes was evaluated by ESI-MS.
primer (23nt)
5’-d(GAC CCA CTC CAT CGA GAT TTC TC)-3’
single stranded template (35nt)
5´-d(GCG CTG GCA CGG GAG AAA TCT CGA TGG AGT GGG TC)-3´.
Farber primer T/C (20nt)
5´-NH2(CH2)6-d(CGT TGG TCC TGA AGG AGG A T/C)-3´
Leiden primer T/C (25nt)
5´-NH2(CH2)6-d(CAA GGA CAA AAT ACC TGT ATT CCT T/C)-3´
DPyD primer T/C (25nt)
5´-NH2(CH2)6-d(GTT TTA GAT GTT AAA TCA CAC TTA T/C)-3´
Farber templates A/G (90nt)
5’-d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC CGC CGT GGA CAG AGG ACT GCA GAA AAT
CAA CCT A/G TC CTC CTT CAG GAC CAA CGT ACA GAG)-3’
Leiden templates A/G (98nt)
5’-d(GAC ATC ATG AGA GAC ATC GCC TCT GGG CTA ATA GGA CTA CTT CTA ATC TGT AAG AGC
AGA TCC CTG GAC AGG C A/G A GGA ATA CAG GTA TTT TGT CCT TG)-3’,
DPyD templates A/G (120nt)
5’-d(AAA GCT CCT TTC TGA ATA TTG AGC TCA TCA GTG AGA AAA CGG CTG CAT ATT GGT GTC
AAA GTG TCA CTG AAC TAA AGG CTG ACT TTC CAG ACA AC A/G TAA GTG TGA TTT AAC ATC
TAA AAC)-3’.
84
Materials and methods
5.19.2 Activation
of
glass
slides
and
spotting
of
amino-modified
oligonucleotides to glass slides
Aminopropyl-silylated
glass
slides
were
derivatized
with
1,4-phenylene
diisothiocyanate (0.2% (w/v)) in a pyridine/dimethylformamide (10% (v/v)) solution
for 2h at room temperature. The slides were subsequently washed several times with
dimethylformamide and acetone, dried under a stream of nitrogen and stored
desiccated until spotting. Spotting of 5’-amino modified primer probes (20 μM), ~4 nl
per spot, in sodium phosphate buffer (150 mM, pH 8.5), was performed between 1922°C and 70-77% humidity. The slide tray was cooled during spotting procedure at
10°C. After the spotting process the slides were incubated at room temperature in a
closed petri dish over a saturated NaCl solution overnight. Thereupon, blocking was
performed in a NH4OH solution (10%) for 30 min, followed by subsequent washing
steps with water. The slides were dried under a stream of nitrogen and stored at 4°C
until further use.
5.19.3 Primer extension and arrayed primer extension
Wild-type and mutant Pfu DNA polymerase was obtained as described72.
The reactions for the primer extension reactions in solution contained 1x Pfu reaction,
dATP, dGTP, dCTP (each 200 μM), single stranded template (200 nM), 5´-[32P]-labelled
primer (150 nM) and Pfu DNA polymerase (100 nM) within an overall volume of 20 μl.
TTP and F3-dUTP concentration was varied (200 μM TTP, 185 μM TTP + 15 μM F3-dUTP
(7.5%), 200 μM F3-dUTP). The reaction mixtures were denatured for 2 min at 95°C,
annealed at 55°C and the reaction was initiated by addition of DNA polymerase (100
nM). After 20 min at 72°C, the primer extension was stopped by addition of gelloading buffer. Product mixtures were separated by denaturing PAGE (12%). Gels were
analysed with a Molecular Imager by phosphor imaging and via Cy3-fluorescence
detection channel.
The arrayed primer elongation reactions contained 1x Pfu reaction buffer, dATP, dGTP,
dCTP (200 μM), TTP (185 μM), F3-dUTP (15 μM), single stranded template (500 nM),
BSA (0.1%), 0.5x self seal reagent and the respective Pfu DNA polymerase (200 nM).
The reaction solution, 3.4 μl per reaction, was placed on the spotted area and covered
85
Materials and methods
with round cover slips (10 mm diameter). Primer extension was carried out with a
microarray thermocycler using the following temperature steps: 95°C for 225 sec.,
55°C for 60 sec. and 72°C for 20 min. The reaction was stopped by cooling the slides to
4°C and the slides were subsequently washed under gentle agitation in 0.1 x SSC
buffer (sodium chloride / sodium citrate) + SDS (0.1%) twice for 10 min and water
three times 5 min. The slides were then dried under a stream of nitrogen directly
before reading out with a GenePix microarray scanner machine.
5.20 Methods for section 2.3
5.20.1 Primers and templates on solid support
abasic site primer-template
p1
5´- NH2C6-CGT TGG TCC TGA AGG AGG AT-3´
t1
5´- AAAACGC TGTAGCA TAG AGT ACATG ACA G TT CX CCT A TC CTC CTT CAG GAC CAA
CG-3´ X = stable abasic site analogue
fully matched primer-template
p2
5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC TTA T-3´
t2
5´-AAA GCT CCT TTC TGA ATA TTG AGC TCA TCA GTG AGA AAA CGG CTG CAT ATT GGT
GTC AAA GTG TCA CTG AAC TAA AGG CTG ACT TTC CAG ACA AC A TAA GTG TGA TTT AAC ATC
TAA AAC-3´
single mismatched primer-template
p3
5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC TTA C-3´
t2
(sequence see above)
distal mismatched primer-template
p4
5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC CTA T-3´
t2
(sequence see above)
86
Materials and methods
triple mismatched primer-template
p5
5´- NH2C6-GTT TTA GAT GTT AAA TCA CA GAA AT-3´
t2
(sequence see above)
surface coverage estimation
p6
5´- NH2C6-CGT TGG TCC TGA AGG AGG AT-biotin-3´
5.20.2 Primers and templates in solution experiments
abasic site primer-template
p1´
5´-CGT TGG TCC TGA AGG AGG AT-3´
t1´
5´-AAA TCA XCC TAT CCT CCT TCA GGA CCA ACG TAC-3´
X = stable abasic site analogue
fully matched primer-template
p2´
5´-GTT TTA GAT GTT AAA TCA CAC TTA T-3´
t2´
5´-CTT TCC AGA CAA CAT AAG TGT GAT TTA ACA TCT AAA AC-3´
single mismatched primer-template
p3´
5´-GTT TTA GAT GTT AAA TCA CAC TTA C-3´
t2´
(sequence see above)
distal mismatched primer-template
p4´
5´- GTT TTA GAT GTT AAA TCA CAC CTA T-3´
t2´
(sequence see above)
triple mismatched primer-template
p5´
5´-GTT TTA GAT GTT AAA TCA CA GAA AT-3´
t2´
(sequence see above)
87
Materials and methods
5.20.3 Overexpression of KlenTaq clones in multiwell format and cell lysate
preparation
KlenTaq clones were parallel expressed in 96x deep-well plates containing 1 ml of LBmedium and 100 μg/ml carbenicillin at 37°C. Overexpression was induced via addition
of 200 μg/l anhydrotetracycline at OD600~0.8 for 4h. Afterwards, the plates were
centrifuged at 4000 x g for 15 min, the supernatants were thrown away and cell pellets
were resuspended in 1x KlenTaq reaction buffer containing an additional amount of
lysozyme (0.1 mg/ml). The sealed plates were subsequently incubated for 15 min at
37°C and then for 40 min at 75°C. After centrifugation at 4000 x g for 45 min the
lysate was directly used for screening.
5.20.4 Spotting of DNA polymerase cell lysate mixtures and screening reactions
Immediatly before spotting, the DNA polymerase cell lysates were mixed 1:1 with a
KlenTaq reaction mix containing 1xKlenTaq buffer, 0.2 % BSA, 400 μM
dATP,dGTP,dCTP, 380 μM dTTP, 20 μM Biotin-11-dUTP and the respective templates in a
96x well plate at 4°C and agitated for 30 sec using a shaker module. The cell lysate
mastermix mixtures were centrifuged for 1 min at 4000 x g. Spotting was then
performed at ~20°C air temperature and air humidity between 60-70%. The slide tray
was also cooled during the whole spotting procedure at 5°C. Cell lysates were spotted
in a way that each lysate covered 2 columns and all 5 different primer spot rows with 3
drops per spot resulting in approx. 1.2 nl total spotting volume. The slides were
subsequently placed in a humidity chamber consisting of a water filled petri dish at
room temperature for ~1 minute until all spots were rehydrated. After incubation, the
slides were shortly rinsed twice under gentle agitation in 0.1 x SSC+SDS buffer (15 mM
NaCl, 15 mM sodium citrate, pH 7.0, 0.01% SDS) for 5 min and twice with water (5 min
each). The slides were then dried under a stream of nitrogen. Afterwards, 70 μl of a
streptavidine-alexa fluor® 546 solution 0.4 μg/ml in 1x TBS-T buffer (10 mM Tris-HCl
pH 8.0 150 mM NaCl, 0.05% Tween-20) was placed on each slide and directly covered
with a square glass cover slips 24 x 60 mm. Incubation was carried out in a humidity
chamber (see Figure 21) at room temperature for another 40 min. The cover slip was
then removed and the slides were rinsed twice with 1x TBS-T buffer and once with
88
Materials and methods
water. Afterwards, the slides were dried under a stream of nitrogen before readout
with a GenePix microarray scanner machine (532 nm channel, 10 μm / pixel
resolution) was performed.
5.20.5 Expression and purification of KlenTaq mutants
KlenTaq wt and mutants were purified in batch technique using Ni-NTA sepharose. In
detail 100 ml cultures were grown to an OD600 of 1.0 at 37°C following induction with
AHT (200 μ g/L) for 3 h. After harvesting cells were lysed for 15 min at 37°C in 5 ml
KlenTaq lysis buffer followed by heat denaturation of host proteins at 75°C for 40 min
and centrifugation at 20.000 x g for 30 min. Supernatants were incubated with Ni-NTA
sepharose slurry. After washing with KlenTaq washing buffer (5 mM and 20 mM
imidazol), elution was carried out using washing buffer (200 mM imidazol). After
buffer exchange to 2x KlenTaq washing buffer, Tween20, (NH4)2SO4 and glycerol was
added to 50 % (see Buffers and solutions, KlenTaq storage buffer). Enzyme purity
(Figure 27) and quantity was controlled and determined by SDS-PAGE using an
albumin standard dilution curve. Purified enzymes were stored at -20°C.
5.20.6 Primer extension reactions in solution
20 μl of the reaction contained 1x KlenTaq buffer, 100 μM dNTPs, 225 nM template,
150 nM 5´-32P-labeled primer. DNA polymerase concentrations were 1 nM for
matched, mismatched and distal mismatched cases and 50 nM for triple-mismatched
and abasic site templates. The reaction mixtures without dNTPs were annealed by
heating for 2 min at 95°C and cooling to 25°C. Reactions were initiated by addition of
dNTPs. After incubation at 51°C for 15 min in cases of match, mismatch and distal
mismatch and 1h for the triple-mismatch and abasic site containing reactions, the
primer extension was stopped by addition of 40 μl of gel-loading buffer (80%
formamide, 20 mM EDTA). Product mixtures were separated by 12% denaturing PAGE
and analysed by phosphorimaging.
89
Materials and methods
5.20.7 Test of the influence of drying and rehydration on the KlenTaq polymerase
activity in solution
Solutions of KlenTaq wt ranging from 400, 40, 4, 0.4, 0.04, 0.004 nM in 1x KlenTaq
reaction buffer were divided in two 10 μl aliquots, respectively. One aliquot was dried
under vacuum using a high-vacuum membrane pump while the other aliquot was
stored at room temperature. After 1 h, the dried residue was dissolved in 10 μl water
and both aliquots were used for subsequent primer extension reactions using fully
matched primer p2´ and template t2´. Reactions (20 μl) contained 1 x KlenTaq buffer,
100 μM dNTPs, 225 nM template, 150 nM 5´-[32P]-labelled primer and 2 μl of the DNA
polymerase containing solution. The reactions were stopped after 15 min incubation at
51°C by addition of 40 μl of gel-loading buffer. The products were separated by 12%
denaturing PAGE and analysed by phosphorimaging (see Figure 22).
5.20.8 Spotting and immobilisation of short DNA oligomers
Activation of glass slides and spotting of amino-modified oligonucleotides to glass
slides were conducted as previously described (see Section 5.19.2)
5.20.9
Estimation of surface coverage of oligonucleotides bound to the chip
surface
To estimate the loading density and the amount of attached DNA primers on the
surface, the resulting fluorescence signal of biotinylated immobilised primers with
directly spotted streptavidin-alexa fluor®546 conjugate were compared: A DNA
oligomer (p6) was immobilised in replicates on a freshly activated aminosilane slide as
described above. After incubation with a streptavidine-alexa fluor®546 solution (0.4
μg/ml in 1x TBS-T buffer (10 mM Tris-HCl pH 8.0 150 mM NaCl, 0.05% Tween-20)) the
slide was washed twice in 1x TBS-T buffer for 2 min. and in water for 10 sec. Afterwards
a dilution series of streptavidine-alexa fluor®546 (1μM – 30nM, 0.4 nl) was spotted
directly neighboured to the DNA replicates. Without additional washing the whole
slide was scanned using a GenePix microarray scanner machine (532 nm channel, 10
μm / pixel resolution). Under the assumption that binding stoichiometry between
90
Materials and methods
streptavidin conjugate to the immobilised biotinylated DNA strand is 1:1, a linear
regression of the fluorescence values obtained from the dilution series made a
calculation of the amount of immobilised DNA possible. The spotted amounts of DNA
polymerase mutants deriving from the cell lysates were determined by SDS-PAGE.
5.21
Methods for section 2.4
5.21.1 Cloning, Protein Expression and Purification of Taq M1 Polymerase
Respective plasmids (pASK-IBA37plus) harbouring the KlenTaq M1 and the Taq wildtype gene were isolated from the respective E. coli cultures using the High Pure
Plasmid Isolation Kit. The KlenTaq M1 polymerase gene28 was amplified by using
Phusion DNA polymerase with the forward primer (5'-GAT CTA CGT CTC CGC CCT GGA GGA
GGC CC-3') and reverse primer (5'-CAG GTC AAG CTT AGT TAG ATA TCA CTC C-3'). The Taq
5´-3´ nuclease domain DNA143 including the whole pASK-IBA37plus plasmid sequence
was amplified by using Phusion DNA polymerase with the forward primer (5'-GCC AAG
GAG TGA TAT CTA ACT AAG CT-3') and reverse primer (5'-ATG ATC CGT CTC AGG GCC TTG
GGG CTT TCC AGA A-3'). Both amplificates were purified by 0.8% agarose gel
electrophoresis and isolated using the QIAquick Gel Extraction Kit. Isolated DNA was
digested by EcoRV and BsmBI, purified using the High Pure PCR Cleanup Micro Kit. The
Taq 5´-3´ nuclease domain amplificate was dephosphorylated using Antarctic
Phosphatase and ligated with the KlenTaq amplificate by using the Rapid DNA Ligation
Kit and transformed into electro competent E. coli XL10 gold cells. Clones were picked
from agar plates and grown separately overnight in LB medium (100 μg/ml
carbenicillin). The integrity of whole mutant clone was proved by sequencing of the
respective purified plasmid (GATC Biotech AG, Germany) using the sequencing primers
p1 - p5 (p1 5’-GAG TTA TTT TAC CAC TCC CT-3’, p2 5’-CCT GGC TTT GGG AAA AG-3’, p3 5’CCC GAG CCT TAT AAA GC-3’, p4 5’-CGT AAG GGA TGG CTA GCT CC-3’, p5 5’-CGC AGT AGC
GGT AAA CG-3’). Enzyme purification and concentration determination was conducted
as described (see Section 5.13)
91
Materials and methods
5.21.2 Nuclease activity assay
Reaction mixtures (60 μl) contained 50 mM Tris•HCl (pH 9.2), 16 mM (NH4)2SO4, 0.1%
Tween 20, 2.5 mM MgCl2, 50 nM of each dNTP, 150 nM substrate DNA (22 nt, 5’-[32P]CCC CCC CCC CTC ATA CGT ACA C-3’, 225 nM template DNA (5’-GTG TAC GTA TGA TCA TGC
AGG TAG CCG ATG AAC TGG TCG AAA GAC CAG TTC ATC GGC TAC CTG CAT GAT-3’). After an
initial denaturation and annealing step (95°C for 5 min, 0.5°C/s cooling down to 4°C),
the reaction mixture was heated to 30°C and the reaction was started by addition of
DNA polymerase (50 nM final concentration). 5 μl aliquots were taken at various time
periods up to 60 min and reaction was stopped by addition of gel loading buffer (80%
formamide, 20 mM EDTA). Product mixtures were analysed by 12 % denaturating PAGE
and quantified using a Phosphorimager.
5.21.3 Real-time PCR, template dilution series
Reaction mixtures (20 μl) contained 50 mM Tris•HCl (pH 9.2), 16 mM (NH4)2SO4, 0.1%
Tween 20, 2.5 mM MgCl2, 250 μM of each dNTP, tenfold dilution series of template RNA
from bacteriophage MS2 (10 nM – 10 fM) or DNA template MS2 (1 nM – 10 fM, 100 nt,
5’-d(ATC GCT CGA GAA CGC AAG TTC TTC AGC GAA AAG CAC GAC AGT GGT CGC TAC ATA GCG
TGG TTC CAT ACT GGA GGT GAA ATC ACC GAC AGC ATG AAG TCC G)-3’), 200 nM of each
primer (5’-d(ATC GCT CGA GAA CGC AAG TT)-3’ forward, 5’-d(CGG ACT TCA TGC TGT CGG
TG)-3’ reverse), 0.6 x SYBRgreenI, 10 nM Taq DNA polymerase wt / M1 and for the
temperature dependence reactions (vide infra) 5 nM enzyme respectively. After an
initial reverse transcription cycle (95°C for 30 s, 55°C for 35 s and 65°C for 30 min) the
product was amplified by 30 PCR cycles (95°C for 30 s, 55°C for 35 s and 72°C for 40 s)
and following melting curve measurement from 30-94°C. In case of DNA templates the
PCR was performed without the RT step. Temperature dependence of reverse
transcriptase activity was tested by applying a temperature gradient (from 60 72°C for
15 min) during the reverse transcription cycle and subsequent amplification as
described above.
92
Materials and methods
5.21.4 Primer extension assay with an RNA template
Reaction mixtures (20 μl) contained 50 mM Tris•HCl (pH 9.2), 16 mM (NH4)2SO4, 0.1%
Tween 20, 2.5 mM MgCl2, 10 nM Taq DNA polymerase wt or M1, 150 nM DNA primer (20
nt, 5’-[32P]-d(CGT TGG TCC TGA AGG AGG AT)-3’), 225 nM template RNA (5’- AAA UCA
ACC UAU CCU CCU UCA GGA CCA ACG-3’). After an initial denaturation and annealing
step (95°C for 2 min, 0.5°C/s cooling to 40°C for 30 s), a temperature gradient (from
60 – 72°C, in detail: 60.1, 60.3, 61.2, 62.5, 63.9, 65.3, 66.7, 68.1, 69.5, 70.8, 71.7,
72.0°C) was applied and the reaction was started by addition of 100 nM dNTPs. After
10 min of incubation the reactions were stopped by addition of gel loading buffer
(80% formamide, 20 mM EDTA). Product mixtures were separated by 12% denaturating
PAGE and visualised using a Phosphorimager.
5.21.5 Real time RT-PCR conditions
Dr. Manfred Weidmann and coworkers, University of Göttingen, performed these
experiments.
Only in short: Real time RT-PCR for Dobrava virus was performed as described145 using
the LightCycler® 480 RNA Master Hydrolysis Probes kit containing an aptamer blocked
Tth. TaqM1 was used using the Tris•HCl-(NH4)2SO4-buffer described above (pH 9.2) or a
bicine buffer (50 mM Bicine (pH 8.2), 115 mM KOAc, 2.5 mM MgCl2, 8% glycerol).
Primer concentrations were 500 nM for the primers and 200 nM for the probe and the
following temperature profile of RT 63°C 5min, Activation 95°C, 1min, 45 cycles of 2step PCR 95°C, 5sec and 60°C 1min was used for both enzymes. A transcribed
quantitative RNA standard was used for sensitivity testing. All tests with the
quantitative RNA standard were done in triplicates.
5.22
Methods for section 2.5
5.22.1 Primer extension assays containing abasic site
Primer was labelled using [γ32P]-ATP according to standard techniques (see Section
5.9). 20 μl of the KlenTaq reactions contained 150 nM primer (5´-d(CGT TGG TCC TGA
AGG AGG ATA GG)-3’), 225 nM abasic site containing template (5’-d(AAA TCA AP*CC TAT
93
Materials and methods
CCT CCT TCA GGA CCA ACG TAC)-3’), 100 μM dNTPs in buffer (50 mM Tris-HCl (pH 9.2), 16
mM (NH4)2SO4, 0.1% Tween 20, 2.5 mM MgCl2) and 50 nM (for experiments using wildtype and DM) and 500 nM (for experiments using mutants Y671A and Y671W) DNA
polymerase. Incubation times are provided in the respective figure legends.
Temperature protocol was as follows: Denaturation at 95°C for 2min, annealing at
30°C for 2 min, followed by incubation at 72°C for certain times as depicted in the
respective figure legends. The reactions were started by addition of only dATP, TTP,
dCTP or dGTP. Reactions were stopped by addition of 40μl stop solution (80 % [v/v]
formamide, 20 mM EDTA, 0.25% [w/v] bromophenol blue, 0.25% [w/v] xylene cyanol)
and analyzed by 12% denaturing PAGE. Visualization was performed by
phosphorimaging.
5.22.2 Primer extension assays containing blunt-ended DNA
Primer was labelled using [γ32P]-ATP according to standard techniques (see Section
5.9). 20 μl of the KlenTaq reactions contained 100 nM primer (5´-d(CGT TGG TCC TGA
AGG AGG AT)-3’), 150 nM template (5’-d(AAA GAA ACC AGG ACT TCC TCC TA)-3’),
respectively, 200 μM dNTPs in buffer (50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 0.1%
Tween 20, 2.5 mM MgCl2) and 100 nM DNA polymerase. Incubation times are provided
in the respective figure legends. Temperature protocol was as follows: Denaturation at
95°C for 2min, annealing at 30°C for 2 min, followed by incubation at 72°C for certain
times as depicted in the respective figure legends. Reactions were started by addition
of only dATP, TTP, dCTP or dGTP. Reactions were stopped by addition of 40 μl stop
solution (80 % [v/v] formamide, 20 mM EDTA, 0.25% [w/v] bromophenol blue, 0.25%
[w/v] xylene cyanol) and analyzed by 12% denaturing PAGE. Visualization was
performed by phosphorimaging.
5.22.3 Site directed mutagenesis
Klentaq (KTQ) wt in pGDR11 vector was purified using High Pure Plasmid Isolation Kit
(Roche) 28,131. Whole plasmid PCR was conducted largely along the QuickChange® SiteDirected Mutagenesis Kit Protocol by Stratagene. For PCR, Phusion DNA Polymerase in
94
Materials and methods
combination with Phusion GC-buffer (provided by the supplier) was used with
following mutagenesis primers:
fwd primer 5'-d(AAC TTC GGG GTC CTC TGG GGC ATG TCG GCC)-3' and rev primer 5'-d(GGC
CGA CAT GCC CCA GAG GAC CCC GAA GTT)-3' for Y671W mutation and fwd primer 5'-d(CTT
CGG GGT CCT CGC CGG CAT GTC GGC C)-3' and rev primer 5'-d(GGC CGA CAT GCC GGC GAG
GAC CCC GAA G)-3' for Y671A mutation. Transformation was performed in E.coli strain
XL10 gold. Sequencing of the open-reading frame was conducted by GATC, Germany
using the following primers: pQE-RP 5´-d(GTT CTG AGG TCA TTA CTG G)-3´, pQE-FP 5´d(CGG ATA ACA ATT TCA CAC AG)-3´, pQE-KTQ-mid 5'-d(CGT AAG GGA TGG CTA GCT
CC)-3'. Ni-NTA purification of over expressed mutants was conducted using standard
protocols (see Section 5.20.5,).
5.22.4 Enzyme kinetics
The rate of single turnover, single nucleotide incorporation was determined using
rapid quench flow kinetics using a chemical quench flow apparatus (RQF-3, KinTek
Corp., University Park, PA). 15 μl of radiolabelled primer/template complex (100 nM)
and DNA polymerase (1 μM) in reaction buffer (20 mM TrisHCl pH 7.5, 50 mM NaCl and
2 mM MgCl2 in case of abasic sites containing experiments and 50 mM Tris-HCl (pH
9.2), 16 mM (NH4)2SO4, 0.1% Tween 20, 2.5 mM MgCl2 in all other experiments) were
rapidly mixed with 15 μl of a dNTP solution in reaction buffer at 37°C. Quenching was
achieved by 0.6% trifluoroacetic acid at defined time intervals. Quenched samples
were analyzed on a 12% denaturing PAGE followed by phosphorimaging. For kinetic
analysis, experimental data were fit by nonlinear regression using the program
GraphPad Prism 4. The data were fit to a single exponential equation:
[conversion]=A·(1-exp(-kobs·t)). The observed catalytic rates (kobs) were then plotted
against the dNTP concentration used and the data were fitted to a hyperbolic equation
kobs=kpol·c(dNTP)/(Kd+c(dNTP)) to determine the Kd of the incoming nucleotide. The
incorporation efficiency is given by kpol/Kd. The kinetic data result from multiple
independently conducted experiments.
95
Abbreviations
6
Abbreviations
A
Adenin
Abb.
Abbildung
Ac
Acetyl
APS
Ammoniumperoxodisulfat
BSA
Bovine serum albumin
C
Cytosin
CD
Cirkular Dichroismus
CMPI
2-Chloro-1-methyl-pyridiniumiodid
CPG
controlled pore glas
DBO
1,4-Diazabicyclo[2,2,2]-octan
DC
Dünnschichtchromatographie
DCM
Dichlormethan
DEAD
Diethylazodicarboxylat
DMAP
4-N,N-Dimethylaminopyridin
DMF
N,N-Dimethylformamid
DMTr
Dimethoxytrityl
DNA
Desoxyribonukleinsäure
EE
Essigsäureethylester
eq.
Äquivalent
h
Stunden
HPLC
high-performance-liquid-chromatography
HV
Hochvakuum
LCAA-CPG
long-chain amino alkyl modified controlled pore glass
LevOH
Levulinsäure
M
molar, [mol / L]
min
Minuten
MS
Mass Spectrometry
NMR
Nuclear Magnetic Resonance
PCR
Polymerase Chain Reaction
96
Abbreviations
PE
Petroleumbenzin
Rf
Retentionsfaktor
RNA
Ribonukleinsäure
RP
reversed phase
sec
Sekunde
T
Thymidin
Tab.
Tabelle
TBAF
Tetra-n-butylammoniumfluorid
TBDMS
tert.-Butyldimethylsilyl
TBE
Tris-Borat-EDTA
TEAA
Triethylammoniumacetat
TEMED
Tetramethylethylendiamin
THF
Tetrahydrofuran
TRIS
Tris-(Hydroxymethyl)aminoethan
UV
ultraviolett
97
Sequences
7
DNA and amino acid sequences
7.1
pASK-IBA37plus ::TaqM1 nucleic acid sequence
ATCTAACTAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTGTCTGCCGTTTA
CCGCTACTGCGTCACGGATCTCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACG
CGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA
CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGC
ACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTT
TCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACC
CTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGA
TTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCAGGTGGCACTTTTCGGGG
AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAAT
AACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCT
TATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGC
TGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAG
TTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC
GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC
ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCAT
GAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTT
GCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA
CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACT
ACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTG
CGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGCTCTCGCGGTAT
CATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGC
AACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAGGAATT
AATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAAT
CGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGT
AAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGC
CCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAA
GTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAA
AATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCAGTGGG
98
Sequences
GCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACC
TACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCA
GCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTT
AAAAGCAGCATAACCTTTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTT
GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGA
TCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA
CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG
CGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC
GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA
CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC
CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC
ACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG
AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTT
ACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATCGAATGGCCAGATGATTAATTC
CTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGA
AATGAATAGTTCGACAAAAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAAATGGCTA
GCAGAGGATCGCATCACCATCACCATCACATCGAAGGGCGCATGAGGGGGATGCTGCCCCTCTTTGAGC
CCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCT
CACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAA
GGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGG
GGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCT
GGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCT
GGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGC
TCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAG
TACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCC
GGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAGTGGGGGAGCCTGGAAGCCC
TCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGA
AGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGG
AGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGG
CCTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTT
GTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTATGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTC
99
Sequences
CACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGAC
CTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCT
CCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGG
CGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAG
AGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGG
GGGTGCGCCTGGACGTGGCCTATCTCAGGGCCATGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCG
AGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCT
CTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGAGCCGC
CGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAA
GCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGC
TTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTC
CGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTG
GACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCC
AGGAGGGGCGGGACTTCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGAC
CCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCT
CCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAA
GGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCG
GCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGGGCGTGCGGGAGGCGGCCGAGCGCATG
GCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCA
GGCTGGGGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAA
GAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCC
CCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGAT
7.2
TaqM1 amino acid sequence
MASRGSHHHHHHIEGRMRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSL
LKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADD
VLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDE
SDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVD
FAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLMALA
AARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARR
YGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRAMSL
100
Sequences
EVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTRAAVLEALREAHPIVEK
ILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEE
GWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDFHTETASWMFGVPREAVDPLMRRAAKTINFGVL
YGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKGV
REAAERMAFNMPVQGTAADLMKLAMVKLFPRLGEMGARMLLQVHDELVLEAPKERAEAVARLAKEV
MEGVYPLAVPLEVEVGIGEDWLSAKE*
Mutations are written in bold letters compared with Taq wild-tpye (L322M, L459M,
S515R, I638F, S739G, E773G)28.
7.3
pASK-IBA37plus ::KlenTaq wt nucleic acid sequence
ATGGCTAGCAGAGGATCGCATCACCATCACCATCACATCGAAGGGCGCGCCCTGGAGGAGGCCCCCTGG
CCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGG
CCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGA
AGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGC
CCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCG
GCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCA
ACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTT
CCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCT
GGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTC
AACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAG
AAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAG
AAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCC
ACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCT
CCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGC
CGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTC
CGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGA
TGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGG
TCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTC
ATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGG
AGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAA
101
Sequences
GAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCAT
GAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCA
CGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTC
ATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTC
CGCCAAGGAGTGATATCTAACTAAGCTTGACCTGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTT
TTTGTCTGCCGTTTACCGCTACTGCGTCACGGATCTCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGG
GTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC
CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGAT
TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC
CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGG
AACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTT
AAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCAGGT
GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG
CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT
TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA
AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT
AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGG
CGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGAC
TTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGT
GCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAG
CTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATG
AAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTA
TTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTTG
CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGT
GGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGA
CGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAG
CATTGGTAGGAATTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA
ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACAT
TGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCA
TACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGAT
GTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATG
AAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTC
102
Sequences
AGCGCAGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAA
AGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAG
GTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGA
AAGTGGGTCTTAAAAGCAGCATAACCTTTTTCCGTGATGGTAACTTCACTAGTTTAAAAGGATCTAGGTG
AAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC
CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAA
AACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGG
CTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC
TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC
GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG
GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT
GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC
AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA
CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA
CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGACCCGACACCATCGAATGGCCAG
ATGATTAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAG
AGAAAAGTGAAATGAATAGTTCGACAAAAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA
CAA
7.4
KlenTaq wt amino acid sequence in pASK-IBA37+
MASRGSHHHHHHIEGRALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKAL
RDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSE
RLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAG
HPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLP
DLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLA
HLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEE
AQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTA
ADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGE
DWLSAKE*
Mutations for the mutants m1, m2 and m3 are written in bold letters. S460P (TCC>CCC) was found for mutant m1, Y455N (TAT->AAT) and V766A (GTG->GCG) for mutant
103
Sequences
m2 and L359P (CTG->CCG), R457G (AGG->GGG), E537G (GAG->GGG), and V586I (GTC>ATC) for mutant m3.
7.5
pGDR11 ::KlenTaq wt nucleic acid sequence
TTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA
ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGT
GCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA
ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA
TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA
ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACAT
TAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCACCTCGAGAAATCATAAAAAATTTATTT
GCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACAATTTCACACAGAATTCATT
AAAGAGGAGAAATTAACTATGAGAGGATCTCACCATCACCATCACCATACGGATCCGCATGCAGCCCTG
GAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGT
GGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAG
CCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAA
GGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCC
CGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCC
GAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAG
GTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATC
TCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCG
GCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGC
CATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGG
CCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACC
CCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCAC
GGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGAT
CCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAG
GGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACAC
GGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAA
GACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACG
104
Sequences
AGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGA
CCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGAC
CTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGG
CACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAG
GATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCC
GGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATA
GGGGAGGACTGGCTCTCCGCCAAGGAGAAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCA
GTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGA
GAATCCAAGCTAGCTTGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGA
TATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATG
TACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAA
GTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTTCGTATGGCAATGAA
AGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACG
TTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGC
GTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATC
CCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCA
TGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTG
TGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGC
GTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGGGTAATGACTCTCTAGCTTGAGGCATCAA
ATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCT
GAGTAGGACAAATCCGCCGCTCTAGATTACCGTGCAGTCGATGATAAGCTGTCAAACATGAGAATTGTG
CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG
TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGT
TTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCA
AGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAAC
ATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGT
AATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTC
ATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT
GAATTTGATTCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGG
GCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCT
TCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTA
GTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGC
105
Sequences
GTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACC
ACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCC
AGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA
ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTC
ACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTC
ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCACCATTCGA
TGGTGTCGGAATTTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCTAGAGCTGCCTCGCGCGT
TTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCG
GATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCAT
GACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTG
AGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTC
TTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCTGTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA
AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCA
GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA
GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT
TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCT
CCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCT
CCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT
GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC
GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGT
ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAA
CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT
CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT
TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT
CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG
ATCTGTCTATTTCGTTCATCCATAGCTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTT
ACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATA
AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTA
ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC
AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA
GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAA
GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA
106
Sequences
GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTG
CTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA
AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTC
GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA
AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA
TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC
AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGAC
ATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCACCTCGAGAAATCATAAAAAATTTAT
TTGCTTTG
7.6
KlenTaq wt amino acid sequence in vector pGDR11
MRGSHHHHHHTDPHAALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKAL
RDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSE
RLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAG
HPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLP
DLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLA
HLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEE
AQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTA
ADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGE
DWLSAKEKA*
Mutation Y671A was introduced by changing codon TAC into GCC by site-directed
mutagenesis. Mutation Y671W was introducted by changing codon TAC into TGG by site
directed mutagenesis (see also Section 5.22.3).
107
References
8
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
108
References
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Watson, J. D. & Crick, F. H. Molecular structure of nucleic acids; a structure for
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Castro, C. et al. Nucleic acid polymerases use a general acid for nucleotidyl
transfer. Nat Struct Mol Biol 16, 212-8 (2009).
Loeb, L. A. & Preston, B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu
Rev Genet 20, 201-30 (1986).
Lehman, I. R., Bessman, M. J., Simms, E. S. & Kornberg, A. Enzymatic synthesis
of deoxyribonucleic acid. I. Preparation of substrates and partial purification of
an enzyme from Escherichia coli. J Biol Chem 233, 163-70 (1958).
Kim, Y. et al. Crystal structure of Thermus aquaticus DNA polymerase. Nature
376, 612-6 (1995).
Li, Y., Korolev, S. & Waksman, G. Crystal structures of open and closed forms of
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Danksagung
Ich werde meine Forschungsjahre in Konstanz immer in äußert positiver Erinnerung halten. Für
meine Promotion waren dafür einige Personen entscheidend verantwortlich und ich möchte
mich hiermit bei diesen bedanken:
Mein Dank kann gar nicht groß genug ausfallen bei Herrn Prof. Dr. Andreas Marx für das immer
spürbare Vertrauen, die Unterstützung in allen erdenklichen Fällen, natürlich für die
Forschungsthemen die ich bearbeiten durfte, die Betreuung der Arbeit, und die wahrhaft
exzellenten Forschungsbedingungen.
Mein Dank gilt auch Herrn Prof. Dr. Wittmann für die Übernahme der Funktion als Koreferent
und mündlicher Prüfer. Der selbige Dank gilt Prof. Dr. Fischer für die Übernahme der Funktion
des 3. Referenten.
Ebenfalls gilt mein Dank Herrn Prof. Dr. Hartig für die Übernahme des Prüfungsvorsitzes.
Bac und Matthias für die kameradschaftliche Männerbastion im Frauenlabor.
Selbstverständlich auch Sabrina und Nadine für die schöne Arbeitsatmosphäre im Labor und die
Rücksichtnahme auf mancherlei Spinnereien.
Markus für die sehr anregenden Gespräche über Forschung und Science Fiction.
Weitere Freunde, die mir unter anderen die Zeit in Konstanz zur schönsten Zeit meines
bisherigen Lebens gemacht haben: Armin, Jutta, Jochen, Georg (Joh!), Henning, Matthias,
Samuel, Sascha, Philipp, Frank S, Frank K., Dominik.
Meine Bachelor- und Masterstudenten, die mir insbesondere zu einigen Publikationen geholfen
haben: Christina, Patrick, Dirk, Nicole, Nina.
Nochmals bei Nina und Matthias, die meine angefangen Forschungsarbeiten exzellent
weiterführen.
Jörg F. für die schöne Kooperation und den spannenden Arbeiten mit PAR.
Vielen Dank an alle weiteren namentlich nicht genannten Arbeitsgruppenmitglieder.
Vlasta für Ihre praktische Unterstützung im Labor.
Die Alumnis nicht zu vergessen:
Christian, Jens, Nick, Francesca, Kathrin, Katharina, Ilka, Thilo, Benni, Jan, Moritz, Günni, und
Heike.
Vielen Dank an Familie Bruske, die mich unterstützen und mich aufgenommen haben wie ein
Familienmitglied.
Vielen Dank, Ellen einfach für Dich und Deine Liebe!
References
9
Eidesstattliche Erklärung
Eidesstattliche Erklärung
Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und
ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus
anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter
Angabe
der
Quelle
gekennzeichnet.
Weitere
Personen,
insbesondere
Promotionsberater, waren an der inhaltlich materiellen Erstellung dieser Arbeit nicht
beteiligt. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder
ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
Konstanz, im August 2009
(Ramon Kranaster)
117