History and Development of the Polymerase Chain
Reaction (PCR)
Copyright 2006 MolecularStation
Polymerase Chain Reaction - PCR
Table of Contents:
Introduction to PCR - Polymerase Chain Reaction
Polymerase Chain Reaction (PCR)
PCR, a Concept to be Discovered
General Principles of the PCR
Polymerases/Reaction Specificity and Efficiency
Utility of PCR
PCR and Molecular Cloning
Misincorporation: Errors of In Vitro Systems
Reaction Specificity
Major Advantages of PCR as a Cloning Method include its
Rapidity, Sensitivity, and Robustness
Limitations of PCR
Instruments for PCR
Oligonucleotide Synthesis
Primer Design
PCR Today
Will PCR ever be replaced? – Helicase-dependent Amplification
(HDA)
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 (Figure 1) 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 process (5,6,8). Cellfree 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 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 b-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 doublestranded 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 (Figure 2). 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 (Figure 3). The end result is
an exponential increase in the total number of DNA fragments that
include the 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 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) (Figure 4) 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 heat-resistant 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 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. (Figure 5)
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
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 nonspecifically 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, nontarget 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 (temperature-sensitive mutant), and is only reactivated 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
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 molecular paleontology 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
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 indiscriminateamplification of
DNA sequences from a source of DNA that is present in extemely
limited quantities. Therefore, although PCR can be applied to ensure
whole genome amplification, it does not have the advantage of cellbased 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 cellbased 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.
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 reannealing 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 exonuclease-free, 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 beta-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'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 twopolymerase 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 (Figure 6). 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 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 kilobases 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 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.
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