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History and Development of the Polymerase
Chain Reaction (PCR)
Introduction to PCR - Polymerase Chain Reaction
More than 30 years ago, the introduction of recombinant DNA technology as
a tool for the biological sciences revolutionized the study of life. Molecular
cloning allowed the study of individual genes of living organisms; however this
technique was dependent on obtaining a relatively large quantity of pure DNA.
This depended on the replication of the DNA of plasmids or other vectors during
cell division of microorganisms (1). Researchers found it extremely laborious
and difficult to obtain a specific DNA in quantity from the mass of genes present
in a biological sample (2). Recombinant DNA technology made possible the first
molecular analysis and prenatal diagnosis of several human diseases. Fetal
DNA obtained by amniocentesis sampling could be analyzed by restriction
enzyme digestion, electrophoresis, southern transfer and hybridization to a
cloned gene or oligonucleotide probes (3). However, southern blotting permitted
only rudimentary mapping of genes in unrelated individuals (4).
Polymerase Chain Reaction (PCR)
PCR, an acronym for Polymerase Chain Reaction (5,6), allowed the production
of large quantities of a specific DNA from a complex DNA template in a simple
enzymatic reaction. PCR is a recently developed procedure for the in vitro
amplification of DNA. PCR has transformed the way that almost all studies
requiring the manipulation of DNA fragments may be performed as a results of its
simplicity and usefulness (7). In the 1980s, Kary Mullis and a team of
researchers at Cetus Corporation at Cetus Corporation conceived of a way to
start and stop a polymerase's action at specific points along a single strand of
DNA. Mullis also realized that by harnessing this component of molecular
reproduction technology, the target DNA could be exponentially amplified. This
DNA amplification procedure was based on an in vitro rather than an in vivo
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process (5,6,8). Cell-free DNA amplification by PCR was able to simplify many of
the standard procedures for cloning, analyzing, and modifying nucleic acids (1).
Previous techniques for isolating a specific piece of DNA relied on gene cloning –
a tedious and slow procedure. PCR, on the other hand Kerry Mullis stated “lets
you pick the piece of DNA you’re interested in and have as much of it as you
want” (2,8). When other Cetus scientists eventually succeeded in making the
polymerase chain reaction perform as desired in a reliable fashion, they had an
immensely powerful technique for providing essentially unlimited quantities of the
precise genetic material molecular biologists and others required for their work
(8). Since the first report in1985, more than 5000 scientific papers were
published by 1992 (1). Furthermore, the large number of publications of course
makes it impossible to review all the important contributions to the development
and application of PCR technology; however we will attempt to review here the
most important developments in the practice of basic PCR.
PCR, a Concept to be Discovered
PCR was thought to be conceived by Dr. Kerry Mullis in 1983 while working
at the Cetus Corporation in Emeryville, CA. However, some pioneering work was
also done by Gobind Khorana in 1971 who described a basic principle of
replicating a piece of DNA using two primers. Progress then was limited by
primer synthesis and polymerase purification issues (9). In Mullis’s head, the
invention grew from a theoretical scheme to perform limited dideoxynucleotide
sequencing of unique human genes using synthetic oligonucleotides for the
purpose of diagnosing common human disease mutations. An obvious obstacle
to such a direct sequencing strategy was the high complexity of the human
genome (3.3 X 109 base pairs). Thus, a second oligonucleotide or primer was
added to block the progression of the synthesis of the first primer. Later
however, this second primer was included to bind to the other DNA strand, so
that each strand of the mutant allele would contribute to the eventual signal. If
the scheme involving simultaneous hybridization of primers to each strand was
modified by heating the mixture and then repeating the annealing and extension
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steps, then the primary signal would be increased even further. Repeating the
steps would enable the products of the first round to be duplicated in the second
cycle, to yield two copies. Repeating the cycle again would result in four copies,
et cetera. Several weeks passed before this great idea was attempted (8). Two
primers were synthesized to be perfectly complementary to each end of the 110
base pair region of a cloned segment of the human β-globin gene, the
amplification was performed, and the products were identified by acrylamide gel
electrophoresis. The end result was the anticipated 110 base pair DNA fragment
and the beginning of PCR as a basic technique in molecular biology (5,6). In
Mullis's original PCR process (5,6,8), the enzyme was used in vitro (in a
controlled environment outside an organism). The double-stranded DNA was
separated into two single strands by heating it to 96°C. At this temperature,
however, the E.Coli DNA polymerase was destroyed so that the enzyme had to
be replenished after the heating stage of each cycle. Mullis's original PCR
process was very inefficient since it required a great deal of time, vast amounts
of DNA-Polymerase, and continual attention throughout the PCR process.
General Principles of the PCR
Examination of the PCR amplification mechanism reveal its simplicity but also
its elegance. Oligonucleotide primers are first designed to be complementary to
the ends of the sequence to be amplified, and then mixed in molar excess with
the DNA template and deoxyribonucleotides in an appropriate buffer. Following
heating to denature the original strands and cooling to promote primer annealing,
the oligonucleotides each bind to a different strand of the target fragment. The
primers are positioned so that when each is extended by the action of a DNA
polymerase, the newly synthesized strands will overlap the binding site of the
opposite oligonucleotide. As the process of denaturation, annealing, and
polymerase extension is continued the primers repeatedly bind to both the
original DNA template and complementary sites in the newly synthesized strands
and are extended to produce new copies of DNA. The end result is an
exponential increase in the total number of DNA fragments that include the
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sequences between the PCR primers, which are finally represented at a
theoretical abundance of 2n, where n is the number of cycles (1,7,13).
Polymerases/Reaction Specificity and Efficiency
A DNA polymerase is a naturally occurring enzyme, a biological
macromolecule that catalyzes the formation and repair of DNA. It works by
binding to a single DNA strand and creating a complementary strand. The
accurate replication of all living matter depends on this activity, where it functions
to duplicate DNA when cells divide (10,11). Only recently have scientists learned
to manipulate this activity and apply it to scientific research. The earliest PCR
experiments utlilized the Klenow fragment of Escherichia coli DNA polymerase I
at a temperature of 37C to amplify specific targets from human genomic DNA
(5,6). Often these PCR reactions produced incompletely pure target product as
judged by gel electrophoresis (1). These initial PCR amplifications with the
Klenow fragment were not highly specific (5,6). Although a unique DNA fragment
could be amplified ~200,000 fold from genomic DNA, only about 1% of the PCR
product was the targeted sequence (13). A specific hybridization probe was
required to analyse the amplified DNA (5,6). Some PCR conditions were
determined to increase the stringency of primer hybridization such as lower
MgCl2 concentrations and higher annealing temperatures. Furthermore, the
concentration of enzyme and primers, the annealing time, extension time, and
number of PCR cycles all were found to effect the specificity of the PCR. Also,
the concentration of a specific sequence in a sample can also influence the
relative homogeneity of the PCR products (1,7,13,14,15). Deoxyribonucleotide
triphosphates and magnesium in an appropriate buffer are also important
ingredients for PCR. The efficiency and specificity of PCRs can be affected by
variations in the concentration and ratio of free magnesium, deoxyribonucleotide
triphosphates, and primers. These reagents must be optimized in order to
achieve high specificity and yield (14). It was also discovered that the effect of
temperature and oligonucleotide primer length on the specificity and efficiency of
amplification by the polymerase chain reaction (15). The inactivation of the
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Klenow fragment of Escherichia coli DNA polymerase I at the high temperature
required for strand separation required the addition of enzyme after the
denaturation step of each cycle (5,6). Prior to 1988, anyone conducting a PCR
reaction procedure was obliged to sit patiently by a series of water baths or
heating blocks and add a fresh aliquot of E.Coli DNA polymerase after each
denaturation step, which was typically carried out by immersing the reaction
vessel in boiling water for ½ a minute to 3 minutes (7). This rather tedious step
was eliminated by the introduction of a thermostable DNA polymerase, the Taq
DNA polymerase (12) once, at the beginning of the PCR reaction. The
thermostable properties of the DNA polymerase activity were isolated from
Thermus aquaticus (Taq) that grow in geysers of over 110C, and have
contributed greatly to the yield, specificity, automation, and utility of the
polymerase chain reaction (1,7,12). The Taq enzyme can withstand repeated
heating to 94C and so each time the mixture is cooled to allow the
oligonucleotide primers to bind the catalyst for the extension is already present
(1,7). However, higher annealing temperatures were not established until the
single “most important development of PCR development” (8), the purification
and commercial distribution of a heat-resistant DNA polymerase from the
thermophilic bacterium Thermus aquaticus (Taq) (12). The isolation of a heatresistant DNA polymerase also allowed primer annealing and extension to be
carried out at elevated temperatures (1,7,12,13), thereby reducing mismatched
annealing to nontarget sequences (non-specific amplification) or increasing
specificity. In this way, for many amplifications the PCR product could be
detected as a single ethidium bromide-stained band on an electrophoretic gel
(12). This increased specificity also increased DNA yield of the target sequence.
Moreover, longer PCR products could be amplified from genomic DNA, probably
due to a reduction in the secondary structure of the template strands at the
elevated temperature used for primer extension. The upper size limit for Klenow
fragment polymerase amplification was only about 400bp. Taq polymerase and
other thermostable polymerases have synthesized fragments up to 10 kb
(1,7,12,13). The availability of Taq polymerase has also greatly simplified the
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automation of the reaction as it is a much easier task to construct an apparatus
that will cycle a reaction tube through different temperatures than to manufacture
a device that would perform both the thermocycling and the addition of enzyme
aliquots. Currently there is a great variety of thermocyclers available
commercially. This development has been a significant factor in the rapid
application of this technology by the scientific community (7).
Utility of PCR
In addition to the production of double-stranded, blunt-ended DNA fragments
which may be formed by PCR, two other features of the PCR scheme contribute
greatly to the utility of PCR. First, the position of binding of the primers defines
the boundaries of the amplified fragment and therefore the prior molecular
cloning requirement of restriction endonuclease recognition sites is not required
for PCR. As only a limited number of DNA sequences are restriction sites, PCR
greatly increases the flexibility of choice of fragment size and composition.
Secondly, it is not necessary for PCR oligonucleotides to be exactly
complementary to the template DNA. “Tails” may be added to the 5’ end of the
primer to introduce sequences within the priming sites which thus may be
exploited to introduce restriction endonuclease recognition sites or other useful
sequences such as mutations into the amplified DNA. This phenomena allowed
the emergence of PCR as a method for rapid DNA cloning (1,7,13).
PCR and Molecular Cloning
Molecular cloning has benefited from the emergence of PCR as a technique.
Direct cloning was first conducted using a 110 bp DNA fragment amplified by
PCR and oligonucleotide primers which contained restriction endonuclease
recognition sites added to their 5’ ends. These sites were used to facilitate
cloning of the amplified DNA into an M13 plasmid (17). The 110 bp fragment was
also sequenced to confirm that this approach was a rapid yet reliable approach to
cloning.
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Misincorporation: Errors of In Vitro Systems
Cell-based DNA cloning involves DNA replication in vivo, which is associated
with a very high fidelity of copying because of proofreading mechanisms.
However, when DNA is replicated in vitro as with PCR, the copying error rate is
considerably greater. The most widely used polymerase, Taq DNA polymerase
however, has no associated 3’to 5’ exonuclease to confer a proofreading
function. Thus the error rate due to base misincorporation during DNA
replication is rather high for Taq: for a 1 kb sequence that has undergone 20
effective cycles of duplication, approximately 40% of the new DNA strands
synthesized by PCR using this enzyme will contain an incorrect nucleotide
resulting from a copying error (16). Therefore, even if the PCR reaction involves
amplification of a single DNA sequence, the final product will be a mixture of
almost matching, but not identical DNA sequences. Despite the errors due to
replication in vitro, DNA sequencing of the total PCR product may give the
correct sequence due to the fact that the incorporation of incorrect bases is
essentially random and the contribution of one incorrect base on one or more
strands is overwhelmed by the contributions from the huge majority of strands
which will have the correct sequence. However, if the PCR product is to be
cloned in cells, several individual clones may need to be sequenced in order to
determine the correct (consensus) sequence, prior to conducting further
experiments. More recently, the problem of infidelity of DNA replication during the
PCR reaction has been considerably reduced by using alternative heat-stable
DNA polymerases which have associated 3’ to 5’ exonuclease activity.
Pyrococcus furiosus (Pfu) DNA polymerases and Thermococcus Litoralis (VENT)
are becoming more widely used because of the proofreading conferred by their
associated 3’ to 5’ exonuclease activity (18). The resulting PCR product of Pfu
for example, has a much lower level of mutations introduced by copying errors:
for a 1 kb segment of DNA that has undergone 20 effective cycles of duplication,
about 3.5% of the DNA strands in the product carry an altered base (16).
Reaction Specificity
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New approaches to improve specificity have been developed based on the
recognition that the Taq DNA polymerase retains considerable enzymatic activity
at temperatures well below the optimum for DNA synthesis. Thus, primers
annealing non-specifically to a partially single stranded template region can be
extended before the reaction reaches 72°C for extension of specifically annealed
primers. If the DNA polymerase is activated only after the reaction has reached
high (>70°C) temperatures, non-target amplification can be minimized (19,20).
This “Hot start” approach can be accomplished by manual addition of an
essential reagent to the selection tube at elevated temperatures. The addition of
ssDNA binding protein has also been reported to increase specific amplification.
A more user friendly approach is to use either inhibition or inactivation of the
DNA polymerase itself. Two types of inhibition of Taq DNA polymerase have
been tried including oligonucleotide inhibition (21) and antibody (22) inhibition.
Highly specific oligonucleotide inhibitors of both Taq DNA polymerases have
been produced. These selectively inhibit DNA polymerase activity at
temperatures below 40°C and have been shown to function in Hot Start
applications. Alternatively, one can use an antibody against Taq DNA
polymerase. The antibody inhibits the DNA polymerase until the temperature of
the PCR is such that the antibody is denatured at a temperature greater than
55°C, thereby releasing the enzyme. However there are disadvantages to this
type of Hot Start conditions. In this case, one needs an antibody for each
different enzyme used in a PCR and for a large number of PCRs this can rise
costs significantly. The most convenient form of Hot Start is to modify the DNA
polymerase in such a way that it is inactive at room temperature (temperaturesensitive mutant), and is only re-activated following incubation at 95°C for 6-15
minutes (23).
Major Advantages of PCR as a Cloning Method include its Rapidity,
Sensitivity, and Robustness
Because of its simplicity, PCR is a popular technique with a wide range of
applications including direct sequencing, genomic cloning, DNA typing, detection
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of infectious microorganisms, site-directed mutagenesis, prenatal genetic
disease research, and analysis of allelic sequence variations (1,7,13,16) which
depend on essentially three major advantages of the method: Speed and ease of
use: DNA cloning by PCR can be performed in a relatively short amount of time,
within a few hours. Usually, a PCR reaction consists of around 30 cycles each
cycle containing a denaturation, synthesis and reannealing step, with an
individual cycle typically taking 3 5 min in an automated thermal cycler. This is
clearly quicker than the time required for cell-based DNA cloning, which could
take weeks of time. Furthermore, it is quite easy to setup a PCR reaction and
the use of a thermocycler machine is also easy. Some time is required for the
design and synthesis of oligonucleotide primers, but this has been simplified by
the availability of computer software for primer design and rapid commercial or
academic synthesis of custom oligonucleotides. Optimization of PCR conditions
may be required such as primer annealing temperature, magnesium
concentration, and primer concentration. However, the creation of gradient PCR
machines which allow a variety of primer annealing temperatures to be tested at
the same time has greatly decreased the time required for this step. Once the
optimal conditions for a reaction have been obtained, the reaction can then be
simply repeated (1,7,13,16). Sensitivity: PCR is capable of amplifying sequences
from minute amounts of target DNA, even the DNA from a single cell (24). Such
exquisite sensitivity has afforded new methods of studying molecular
pathogenesis and has found numerous applications in forensic science, in
diagnosis, in genetic linkage analysis using single-sperm typing and in
molecularpaleontology studies, where samples may contain minute numbers of
cells. However, the extreme sensitivity of the method means that great care has
to be taken to avoid contamination of the sample under investigation by external
DNA, such as from minute amounts of cells from the operator (1,7,13,16).
Robustness: A broad range of nucleic acid sources are suitable templates for
PCR amplification. Purified DNAs from various species and sources have been
amplified. PCR can permit amplification of specific sequences from material in
which the DNA is badly degraded or embedded in a medium from which
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conventional DNA isolation is problematic. As a result, it is again very suitable for
molecular anthropology and paleontology studies, for example the analysis of
DNA recovered from archaeological remains. It has also been used successfully
to amplify DNA from formalin-fixed or paraffin-embedded tissue samples, which
has important applications in molecular pathology and, in some cases, genetic
linkage studies. Generally, the success of PCR amplification is greatest when
target fragments are relatively abundant (1,7,13,16).
Limitations of PCR
Despite its huge popularity, PCR has certain limitations as a method for
selectively cloning specific DNA sequences. In order to construct specific
oligonucleotide primers that permit selective amplification of a particular DNA
sequence, some prior sequence information is usually necessary. This normally
means that the DNA region of interest has been partly characterized previously,
often following prior cell-based DNA cloning. However, a variety of approaches
have been developed that reduce or even exclude the need for prior DNA
sequence information concerning the target DNA. Previously uncharacterized
DNA sequences can sometimes be cloned using PCR with degenerate
oligonucleotides if they are members of a gene or repetitive DNA family at least
one of whose members has previously been characterized. In some cases,
PCR can be used effectively without any prior sequence information concerning
the target DNA to permit indiscriminate amplification of DNA sequences from a
source of DNA that is present in extremely limited quantities. Therefore,
although PCR can be applied to ensure whole genome amplification, it does not
have the advantage of cell-based DNA cloning in offering a way of separating the
individual DNA clones comprising a genomic DNA library. The amount of PCR
product obtained in a single reaction is also much more limited than the amount
that can be obtained using cell-based cloning where scale-up of the volumes of
cell cultures is possible. The efficiency of a PCR reaction will vary from template
to template and according to various factors that are required to optimize the
reaction but typically only comparatively small amounts of product are achieved.
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Although the theoretical yield of PCR is exponential, the actual yield of a PCR is
much less indicating that the scheme is operating with less than its maximum
potential. For example, the amount of product at each cycle eventually levels off.
This plateau may be explained by the following phenomena. First, some of the
template may never be available due to strand breaks or failure of the DNA to
dissociated from other macromolecules during purification and the initial
thermocycles. Secondly, the amount of enzyme is finite and eventually activity
may decrease. Thirdly, as the concentration of the double-stranded product
reaches high levels, competition increases between annealing of template (PCR
product) to primer and re-annealing of the complementary template strands
(1,7,13). An obvious and many times great disadvantage of PCR as a DNA
cloning method has been the size range of the DNA sequences that can be
cloned. Unlike cell-based DNA cloning where the size of cloned DNA sequences
can approach 2 Mb, reported DNA sequences cloned by PCR have typically
been in the 0.1 5 kb size range, often at the lower end of this scale. Small
fragments of DNA can usually be amplified easily by PCR, however it becomes
increasingly more difficult to obtain efficient amplification as the desired product
length increases. Barnes (25) recognized a target length limitation to PCR
amplification of DNA. He used a combination of a high level of an exonucleasefree, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very
low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity
(Pfu, Vent, or Deep Vent) to conduct high fidelity long PCR. At least 35 kb of
bacteriophage lambda can be amplified to high yields from 1 ng of lambda DNA
template. Use of this method yielded increased base-pair fidelity, the ability to
use PCR products as primers, and the maximum yield of target fragment. Other
conditions have been identified for effective amplification of longer targets,
including amplification of up to 22 kb of the β-globin gene cluster from human
genomic DNA and up to 42 kb from phaga lambda DNA (26). The conditions for
these long PCRs included increased pH, addition of glycerol and dimethyl
sulfoxide, decreased denaturation times, increased extension times, and the use
of a secondary thermostable DNA polymerase that possesses a 3'-to 5'-
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exonuclease, or "proofreading," activity. The "long PCR" protocol maintained the
specificity required for targets in genomic DNA by using lower levels of
polymerase and temperature and salt conditions for specific primer annealing.
The ability to amplify DNA sequences of 10-40 kb will bring the speed and
simplicity of PCR to genomic mapping and sequencing and facilitate studies in
molecular genetics (26). Generally, the conditions for long range PCR involve a
combination of modifications to standard conditions with a two- polymerase
system. This provides optimal levels of DNA polymerase and 3’to 5’
exonuclease activity which serves as a proofreading mechanism (16).
Instruments for PCR
Thermocyclers which automatically regulate temperatures for PCR cycling
were introduced in 1986. In addition to the advances in PCR reagents, new
instruments for automated thermal cycling and for analyzing PCR products have
been developed. New thermal cyclers have increased rates of heating, cooling,
and heat transfer to modified reaction vessels. The reaction vessels
accommodated by the first generation thermal cyclers (or even water baths and
heating blocks) were standard plastic microfuge tubes. PCR amplification in thin
capillary tubes allowed rapid thermal cycling, and DNA synthesis to 20s. The
speed of the temperature changes achieved in these systems has allowed the
precise definition of temperature optima for each individual step in the PCR
cycle. The new generation thermal cyclers also accommodate more samples,
have more precise thermal profiles, and are programmable (13).
Oligonucleotide Synthesis
One of the least appreciated contributions to the widespread application of
PCR has been the development of reliable automated chemistry for
oligonucleotide synthesis. Until recently, the construction of a single
oligonucleotide was a substantial task that could only be performed by a skilled
organic chemist. Now it is possible to purchase either an oligonucleotide
synthesizer that can be operated by a technician or the oligonucleotides
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themselves from a commercial or academic source. Multiplex oligonucleotide
synthesis machines have been constructed with the aim of reducing the overall
cost of synthesis (27,28). As the oligonucleotides define the eventual PCR
products, there is little doubt that in the absence of their ready supply, PCR
would not have enjoyed the wide acceptance that it has gained today (13).
Primer Design
Researchers agreed early on that the design of PCR primers was difficult and
unreliable. Computer programs were devised to take all of the design criteria into
account. One of the first programs written for primer design was Olga which
made use of the implementation of Digital Research GEM (Graphics
Environment Manager) on the Atari ST (29). Olga was specifically suited to the
polymerase chain reaction (PCR) allowing simultaneous analysis of two primer
sequences. The advantage of Olga was that it provided in one program analyses
for direct repeats, secondary structures and primer dimerization as well as
several useful 'finishing' tools for workers engaged in PCR optimization and
oligonucleotide syntheses. The Primer3 program at the Whitehead Institute is
now thought to be the most reliable and versatile tool currently available (30).
PCR Today
PCRs can now be performed enabling the amplification of DNA fragments up to
several kilo bases in length by more than one million times their initial
abundance. The procedure is highly automatable and requires just a few hours
from beginning the thermocyling to product analysis. This was not the case
previously, and the practical requirements for performing a PCR have been
greatly simplified since the first manuscripts of the method (13). Today, most of
the initial hitches or inefficiencies of the PCR have been worked out (8).
Furthermore, PCR has expanded to include more than 270,000 articles (31).
Will PCR ever be replaced? – Helicase-dependent Amplification (HDA)
Polymerase chain reaction is the most widely used method for in vitro DNA
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amplification however it requires thermal denaturation or thermocycling to
separate the two DNA strands. In vivo, DNA is replicated by DNA polymerases
with various accessory proteins. DNA helicase, a DNA polymerase accessory
proteins acts to separate duplex DNA inside cells. Vincent et al. (32) have
devised a new in vitro isothermal DNA amplification method by mimicking the in
vivo replication mechanism. Helicase-dependent amplification (HDA) utilizes a
DNA helicase to generate single-stranded templates for primer hybridization.
Subsequent primer extension is then catalyzed by a DNA polymerase. HDA
does not require an expensive thermocycler and thus PCR may be performed
practically anywhere. In addition, it offers several advantages over other
isothermal DNA amplification methods by having a simple reaction scheme and
being a true isothermal reaction that can be performed at one temperature for the
entire process. HDA offers great promise in the development of simple portable
DNA diagnostic devices to be used in the field and at the point-of-care (32).
Conclusions
It is said the simplest and most convenient way to define PCR is as a
technique. However, such a categorization eliminates the history of PCR's
development as many individuals over the years contributed to the ideas behind
the theory of PCR and the fine-tuning of the technique. The next simplest
answer is to name an individual as the inventor of the polymerase chain reaction.
Karry Mullis was awarded the Nobel Prize for Chemistry in 1993 for his discovery
of PCR. However, this discovery is contested amongst many scientists, all of
which may have contributed to unlocking this puzzle. It has also been said that
PCR did not exist until it was made to work in an experimental system. With this
in mind, merely the thought of a concept is not sufficient; a concept must have
been successfully been put into practice (33). Although there is doubt as to the
ultimate creator of PCR, and doubt as to the possibility that PCR may somehow
or sometime be replaced, there is little doubt the impact that PCR has created
over a short time span on the study of molecular biology and life.
References
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