2
Application of Restriction Enzymes
2.1
Detection of DNA Fragments
Separation of DNA fragments occurs in
Agarose gels (0.3 – 2%): 300 bp – >1 Mbp
Polyacrylamide gels: 1 – 1000 bp
Agarose
Solubilization at 92°C
Solidifying at 42°C
Low-melting point agarose:
Solubilization at 62°C
Solidifying at 25°C
Preparation of Polyacrylamide
Electrophoresis Through a Gel
Separates DNA and RNA Molecular
According To Size
ƒ Pores in the gel matrix sieve
the DNA molecules according
to their volume
ƒ The large molecules move
slower
ƒ After a given time, molecule
of different size are separated
because they have moved
different distances
ƒ Circular<linear<supercoiled
Electrophoretic
Separation of Linear
DNA Fragments
How to visualize the DNA
fragments ?
Ethidium Bromide
Intrinsic fluorescence: weak
Fluorescence after intercalation: strong
Detection limit: ~ 5 ng DNA per band
Irradiation at 254 nm or 320 nm (1/10)
Energy is re-emitted at 590 nm
Works also with ssDNA and RNA (1/10)
Electrophoretic
Separation of Linear
DNA Fragments
How to determine the size
of these fragments ?
Electrophoretic Migration Pattern of
Double-Stranded DNA
2.2
Pulse-Field Gel Electrophoresis
(PFGE)
Separation of large DNA fragments: 20 kb - > 1
Mbp
Principle:
Alternating electric fields with different pulse
lengths
Electrode Configuration of FIGE (Field
Inversion)
Electrode Configuration of PFGE (PulsedField Gel Electrophoresis)
Electrode Configuration of CHEF (ContourClamped Homogeneous Electric Field)
Electrode Configuration of OFAGE
(Orthogenal-Field Alternation)
Separation of the 16 Yeast
Chromosomes
2.3
Establishment of Restriction
Maps
2.3.1
The Smith-Birnstiel Method
The Different Steps of the SmithBirnstiel Procedure:
1. Identification of a unique restriction site
within the recombinant plasmid (homing endon.)
2. Linearization of the recombinant plasmid
3. Radioactive labeling at one or both ends
4. If labeled at both ends, cleavage with a second
enzyme, separation of the two fragments
5. Partial digestion, separation of the fragments,
autoradiography
The Smith-Birnstiel Mapping
Procedure
partial digestion
autoradiography
2.3.2
Restriction Maps of
Recombinant λ Phages and
Cosmids
Principle of Restriction Fragment Mapping
within Recombinant λ DNA Molecules
cos
2.4
Cloning of Restriction Fragments
Principle of DNA Cloning
2.5
Cloning Without Restriction
Enzymes and DNA Ligase
Seamless Cloning and Gene Fusion
Definition:
Processes that allow two or more DNA
fragments to be joined precisely so that no
unwanted nucleotides are added at the
junctions between DNA fragments
Seamless cloning does not require neither
restriction enzymes nor DNA ligase
Q Lu (2005) Trends Biotechnol. 23: 199
The In-Fusion Cloning Protocol
http://www.clontech.com/products/detail.a
sp?product_id=162275&tabno=2
The Gateway Cloning System
Cloning is independent of restriction enzymes
and DNA ligase
att: core recombination
site is 21 bp
ccdB: toxin; inactivates
DNA gyrase
ccdA: antidot
D Esposito (2009) Methods Mol. Biol. 498: 31
Ligation-Independent Cloning of
PCR Fragments (LIC-PCR)
C Aslanidis (1990) Nucleic Acids Res. 18: 6069