Polymerase chain reaction
“PCR” redirects here. For other uses, see PCR (disambiguation).
A strip of eight PCR tubes, each tube contains a 100μl reaction.
The polymerase chain reaction (PCR) is a biochemistry and molecular biology
technique[1] for exponentially amplifying DNA, via enzymatic replication, without
using a living organism (such as E. coli or yeast). As PCR is an in vitro technique, it
can be performed without restrictions on the form of DNA, and it can be extensively
modified to perform a wide array of genetic manipulations.
Invented in 1983 by Kary Mullis, PCR is now a common technique used in medical
and biological research labs for a variety of tasks, such as the detection of hereditary
diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases,
the cloning of genes, paternity testing, and DNA computing.
Contents
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
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1 PCR in practice
o 1.1 Procedure
o 1.2 PCR optimization
o 1.3 Practical modifications to the PCR technique
o 1.4 Recent developments in PCR techniques
2 Uses of PCR
o 2.1 Genetic fingerprinting
o 2.2 Paternity testing
o 2.3 Detection of hereditary diseases
o 2.4 Cloning genes
o 2.5 Mutagenesis
o 2.6 Analysis of ancient DNA
o 2.7 Genotyping of specific mutations
o 2.8 Comparison of gene expression
o 2.9 AIDS testing
3 History


o 3.1 Patent wars
4 References
5 External links
PCR in practice
Figure 1: A thermal cycler for PCR
PCR is used to amplify specific regions of a DNA strand. This can be a single gene,
just a part of a gene, or a non-coding sequence. Most PCR methods typically amplify
DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for
amplification of fragments up to 40 kb in size.[2]
PCR, as currently practiced, requires several basic components [3]. These components
are:



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


DNA template that contains the region of the
DNA fragment to be amplified
One or more primers, which are complementary
to the DNA regions at the 5' and 3' ends of the
DNA region that is to be amplified.
a DNA polymerase (e.g. Taq polymerase or
another DNA polymerase with a temperature
optimum at around 70°C), used to synthesize a
DNA copy of the region to be amplified
Deoxynucleotide triphosphates, (dNTPs) from
which the DNA polymerase builds the new
DNA
Buffer solution, which provides a suitable
chemical environment for optimum activity and
stability of the DNA polymerase
Divalent cation, magnesium or manganese ions;
generally Mg2+ is used, but Mn2+ can be utilized
for PCR-mediated DNA mutagenesis, as higher
Mn2+ concentration increases the error rate
during DNA synthesis [4]
Monovalent cation potassium ions
The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a
reaction volume typically of 15-100 μl, that are inserted into a thermal cycler. This is
a machine that heats and cools the reaction tubes within it to the precise temperature
required for each step of the reaction. Most thermal cyclers have heated lids to
prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of
oil may be placed on the reaction mixture to prevent evaporation.
[edit] Procedure
Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2)
Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here.
The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is
carried out in three steps (Fig. 2), often preceded by one temperature hold at the start
and followed by one hold at the end.
1. Prior to the first cycle, during an initialization
step, the PCR reaction is often heated to a
temperature of 94-96°C (or 98°C if extremely
thermostable polymerases are used), and this
temperature is then held for 1-9 minutes. This
first hold is employed to ensure that most of the
DNA template and primers are denatured, i.e.,
that the DNA is melted by disrupting the
hydrogen bonds between complementary bases
of the DNA strands. Also, some PCR
polymerases require this step for activation (see
hot-start PCR).[5] Following this hold, cycling
begins, with one step at 94-98°C for 20-30
seconds (denaturation step).
2. The denaturation is followed by the annealing
step. In this step the reaction temperature is
lowered so that the primers can anneal to the
single-stranded DNA template. Brownian
motion causes the primers to move around, and
DNA-DNA hydrogen bonds are constantly
formed and broken between primer and
template. Stable bonds are only formed when
the primer sequence very closely matches the
template sequence, and to this short section of
double-stranded DNA the polymerase attaches
and begins DNA synthesis. The temperature at
this step depends on the melting temperature of
the primers, and is usually between 50-64°C for
20-40 seconds.
3. The annealing step is followed by an
extension/elongation step during which the
DNA polymerase synthesizes new DNA strands
complementary to the DNA template strands.
The temperature at this step depends on the
DNA polymerase used. Taq polymerase has a
temperature optimum of 70-74°C; thus, in most
cases a temperature of 72°C is used. The
hydrogen bonds between the extended primer
and the DNA template are now strong enough
to withstand forces breaking these attractions at
the higher temperature. Primers that have
annealed to DNA regions with mismatching
bases dissociate from the template and are not
extended. The DNA polymerase condenses the
5'-phosphate group of the dNTPs with the 3'hydroxyl group at the end of the nascent
(extending) DNA strand, i.e., the polymerase
adds dNTP's that are complementary to the
template in 5' to 3' direction, thus reading the
template in 3' to 5' direction. The extension time
depends both on the DNA polymerase used and
on the length of the DNA fragment to be
amplified. As a rule-of-thumb, at its optimum
temperature, the DNA polymerase will
polymerize a thousand bases in one minute. A
final elongation step of 5-15 minutes
(depending on the length of the DNA template)
after the last cycle may be used to ensure that
any remaining single-stranded DNA is fully
extended. A final hold of 4-15°C for an
indefinite time may be employed for short-term
storage of the reaction, e.g., if reactions are run
overnight.
Figure 3: Ethidium bromide-stained PCR products after Gel electrophoresis. Two sets
of primers were used to amplify the IGF gene from three different DNA samples. In
sample #1 the gene was not amplified by PCR, whereas bands for tissue #2 and #3
indicate successful amplification of the IGF gene. A positive control, and a DNA
ladder containing DNA fragments of defined length (last lane to the right) to estimate
fragment sizes in the experimental PCRs, were also ran on this gel
To check whether the PCR generated the anticipated DNA fragment (also sometimes
referred to as amplimer), agarose gel electrophoresis is commonly employed for size
separation of the PCR products. The size(s) of PCR products is thereby determined by
comparison with a DNA ladder, which contains DNA fragments of known size, ran on
the gel alongside the PCR products (see Fig. 3).
PCR optimization
Main article: PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to
contamination causing amplification of spurious DNA products. Because of this, a
number of techniques and procedures have been developed for optimizing PCR
conditions. Contamination with extraneous DNA is addressed with lab protocols and
procedures that separate pre-PCR reactions from potential DNA contaminants.[3] This
usually involves spatial separation of PCR-setup areas from areas for analysis or
purification of PCR products, and thoroughly cleaning the work surface between
reaction setups. Primer-design techniques are important in improving PCR product
yield and in avoiding the formation of spurious products, and the usage of alternate
buffer components or polymerase enzymes can help with amplification of long or
otherwise problematic regions of DNA.
[edit] Practical modifications to the PCR technique

Nested PCR - Nested PCR increases the
specificity of DNA amplification, by reducing
background due to non-specific amplification of
DNA. Two sets of primers are being used in
two successive PCR reactions. In the first
reaction, one pair of primers is used to generate
DNA products, which besides the intended
target, may still consist of non-specifically
amplified DNA fragments. The product(s)
(sometimes after gel purification after
electrophoresis of the PCR product) are then
used in a second PCR reaction with a set of
primers whose binding sites are completely or
partially different from the primer pair used in
the first reaction, but are completely within the
DNA target fragment. Nested PCR is often
more successful in specifically amplifying long
DNA fragments than conventional PCR, but it
requires more detailed knowledge of the target
sequences.
 Intersequence specific (ISSR) PCR
 Ligation-mediated PCR

Inverse PCR - Inverse PCR is a method used to
allow PCR when only one internal sequence is
known. This is especially useful in identifying
flanking sequences to various genomic inserts.
This involves a series of DNA digestions and
self ligation, resulting in known sequences at
either end of the unknown sequence.

RT-PCR - RT-PCR (Reverse Transcription
PCR) is a method used to amplify, isolate or
identify a known sequence from a cellular or
tissue RNA. The PCR reaction is preceded by a
reaction using reverse transcriptase to convert
RNA to cDNA. RT-PCR is widely used in
expression profiling, to determine the
expression of a gene or to identify the sequence
of an RNA transcript, including transcription
start and termination sites and, if the genomic
DNA sequence of a gene is known, to map the
location of exons and introns in the gene. The 5'
end of a gene (corresponding to the
transcription start site) is typically identified by
a RT-PCR method, named RACE-PCR, short
for Rapid Amplification of cDNA Ends.
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Assembly PCR - Assembly PCR is the
completely artificial synthesis of long gene
products by performing PCR on a pool of long
oligonucleotides with short overlapping
segments. The oligonucleotides alternate
between sense and antisense directions, and the
overlapping segments serve to order the PCR
fragments so that they selectively produce their
final product.

Asymmetric PCR - Asymmetric PCR is used to
preferentially amplify one strand of the original
DNA more than the other. It finds use in some
types of sequencing and hybridization probing
where having only one of the two
complementary stands is required. PCR is
carried out as usual, but with a great excess of
the primers for the chosen strand. Due to the
slow (arithmetic) amplification later in the
reaction after the limiting primer has been used
up, extra cycles of PCR are required. A recent
modification on this process, known as LinearAfter-The-Exponential-PCR (LATE-PCR),
uses a limiting primer with a higher melting
temperature (Tm) than the excess primer to
maintain reaction efficiency as the limiting
primer concentration decreases mid-reaction.

Quantitative PCR - Q-PCR (Quantitative PCR)
is used to measure the quantity of a PCR
product (preferably real-time). It is the method
of choice to quantitatively measure starting
amounts of DNA, cDNA or RNA. Q-PCR is
commonly used to determine whether a DNA
sequence is present in a sample and the number
of its copies in the sample. The method with
currently the highest level of accuracy is
Quantitative real-time PCR. It is often
confusingly known as RT-PCR (Real Time
PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are
more appropriate contractions. RT-PCR
commonly refers to reverse transcription PCR
(see above), which is often used in conjunction
with Q-PCR. QRT-PCR methods use
fluorescent dyes, such as Sybr Green, or
fluorophore-containing DNA probes, such as
TaqMan, to measure the amount of amplified
product in real time.

Touchdown PCR - Touchdown PCR is a
variant of PCR that aims to reduce nonspecific
background by gradually lowering the
annealing temperature as PCR cycling
progresses. The annealing temperature at the
initial cycles is usually a few degrees above the
Tm of the primers used, while at the later cycles,
it is a few degrees below the primer Tm. The
higher temperatures give greater specificity for
primer binding, and the lower temperatures
permit more efficient amplification from the
specific products formed during the initial
cycles.

Hot-start PCR is a technique that reduces nonspecific amplification during the initial set up
stages of the PCR. The technique may be
performed manually by simply heating the
reaction components briefly at the melting
temperature (e.g., 95˚C) before adding the
polymerase.[6] Specialized enzyme systems
have been developed that inhibit the
polymerase's activity at ambient temperature,
either by the binding of an antibody[5] or by the
presence of covalently bound inhibitors that
only dissociate after a high-temperature
activation step. Hot-start/cold-finish PCR is
achieved with new hybrid polymerases that are
inactive at ambient temperature and are
instantly activated at elongation temperature.

Colony PCR - Bacterial clones (E.coli) can be
rapidly screened for correct DNA vector
constructs. Selected bacterial colonies are
picked with a sterile toothpick from an agarose
plate and dabbed into the master mix or sterile
water. Primers (and the master mix) are added,
and the PCR is started with an extended time at
95˚C when standard polymerase is used or with
a shortened denaturation step at 100˚C and
special chimeric DNA polymerase.[7]

Multiplex-PCR - The use of multiple, unique
primer sets within a single PCR reaction to
produce amplicons of varying sizes specific to
different DNA sequences. By targeting multiple
genes at once, additional information may be
gained from a single test run that otherwise
would require several times the reagents and
more time to perform. Annealing temperatures
for each of the primer sets must be optimized to
work correctly within a single reaction, and
amplicon sizes, i.e., their base pair length,
should be different enough to form distinct
bands when visualized by gel electrophoresis.

Methylation Specific PCR - Methylation
Specific PCR (MSP) is used to detect
methylation of CpG islands in genomic DNA.
DNA is first treated with sodium bisulfite,
which converts unmethylated cytosine bases to
uracil, which is recognized by PCR primers as
thymine. Two PCR reactions are then carried
out on the modified DNA, using primer sets
identical except at any CpG islands within the
primer sequences. At these points, one primer
set recognizes DNA with cytosines to amplify
methylated DNA, and one set recognizes DNA
with uracil or thymine to amplify unmethylated
DNA. MSP using qPCR can also be performed
to obtain quantitative rather than qualitative
information about methylation.
Recent developments in PCR techniques

A more recent method which excludes a
temperature cycle, but uses enzymes, is
helicase-dependent amplification.
 TAIL-PCR, developed by Liu et al. in 1995, is
the thermal asymmetric interlaced PCR.
 Meta-PCR, developed by Andrew Wallace,
allows to optimize amplification and direct
sequence analysis of complex genes. Details at
National Genetic Reference Laboratory,
Manchester, UK
 Multiplex Ligation-dependent Probe
Amplification (MLPA) permits multiple targets
to be amplified with only a single primer pair,
thus avoiding the resolution limitations of
multiplex PCR.
Uses of PCR
PCR can be used for a broad variety of experiments and analyses. Some examples are
discussed below.
Genetic fingerprinting
Genetic fingerprinting is a forensic technique used to identify a person by comparing
his or her DNA with the DNA in a given sample. An example is blood from a crime
scene whose DNA is being genetically compared to DNA from a suspect. The sample
may contain only a very small amount of DNA (obtained from a source such as blood,
semen, saliva, hair, or other DNA-containing organic material). With the use of PCR,
in theory, only a single DNA strand is needed, providing very high sensitivity to this
technique, but increasing the risk of confounding results due to possible
contamination with, and amplification of, DNA from extraneous sources. There are
different PCR-based methods for fingerprinting, summarized in Genetic
fingerprinting. The overall pattern of PCR-generated DNA fragments after gel
electrophoresis and visualization by ethidium bromide staining (or hybridization with
a DNA probe after Southern blotting), can be considered a DNA fingerprint analogous
to the fingerprint pattern unique to each individual.
] Paternity testing
Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child.
(3) Mother. The child has inherited some, but not all of the fingerprint of each of its
parents, giving it a new, unique fingerprint.
Although these resulting 'fingerprints' are unique , genetic relationships, for example,
parent-child or siblings, can be determined from two or more genetic fingerprints,
which can be used for paternity tests (Fig. 4). A variation of this technique can also be
used to determine evolutionary relationships between organisms.
Detection of hereditary diseases
The detection of hereditary diseases in a given genome is a long and difficult process,
which can be shortened significantly by using PCR. Each gene in question can easily
be amplified through PCR by using the appropriate primers and then sequenced to
detect mutations.
Viral diseases, too, can be detected using PCR through amplification of the viral
DNA. This analysis is possible right after infection, which can be from several days to
several months before actual symptoms occur. Such early diagnoses give physicians a
significant lead in treatment.
Cloning genes
Cloning a gene, not to be confused with cloning a whole organism, describes the
process of isolating a gene from one organism and then inserting it into another
organism (now termed a genetically modified organism (GMO)). PCR is often used to
amplify the gene, which can then be inserted into a vector (a vector is a piece of DNA
which 'carries' the gene into the GMO) such as a plasmid (a circular DNA molecule)
(Fig. 5). The DNA can then be transferred into an organism (the GMO) where the
gene and its product can be studied more closely. Expressing a cloned gene (when a
gene is expressed the gene product (usually protein or RNA) is produced by the
GMO) can also be a way of mass-producing useful proteins, for example medicines or
the enzymes in biological washing powders. The incorporation of an affinity tag on a
recombinant protein will generate a fusion protein which can be more easily purified
by affinity chromatography.
Figure 5: Cloning a gene using a plasmid.
(1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple copies of a single gene
from organism A. (4) Insertion of the gene into a plasmid. (5) Plasmid with gene from
organism A. (6) Insertion of the plasmid in organism B. (7) Multiplication or
expression of the gene, originally from organism A, occurring in organism B.
Mutagenesis
Mutagenesis is a way of introducing changes to the sequence of nucleotides in the
DNA. There are situations in which researchers are interested in mutated (changed)
copies of a given DNA strand, for example, when trying to assess the function of a
particular nucleotide sequence within a gene, intergenic regulatory sequence, or for in
in-vitro protein evolution (also known as Directed evolution). Mutations can be
introduced into DNA sequences in two fundamentally different ways in the PCR
process. Site-directed mutagenesis allows the experimenter to introduce a mutation at
a specific location in the DNA strand. Usually, the desired mutation is incorporated in
the primers used in the PCR. Random mutagenesis, on the other hand, is based on the
use of error-prone polymerases or addition of specific ions (such as Mn2+) to the PCR.
In random mutagenesis, the location and nature of the mutations cannot be controlled.
One application of random mutagenesis is to analyze structure-function relationships
of a protein. By randomly altering a DNA sequence, one can compare the resulting
protein with the original and determine the function of each part of the protein.
Analysis of ancient DNA
Using PCR, it becomes possible to analyze DNA that is thousands of years old. PCR
techniques have been successfully used on animals, such as a forty-thousand-year-old
mammoth, and also on human DNA, in applications ranging from the analysis of
Egyptian mummies to the identification of a Russian Tsar[8].
Genotyping of specific mutations
Through the use of allele-specific PCR, one can easily determine which allele of a
mutation or polymorphism an individual has. Here, one of the two primers is
common, and would anneal a short distance away from the mutation, while the other
anneals right on the variation. The 3' end of the allele-specific primer is modified, to
only anneal if it matches one of the alleles. If the mutation of interest is a T or C
single nucleotide polymorphism (T/C SNP), one would use two reactions, one
containing a primer ending in T, and the other ending in C. The common primer
would be the same. Following PCR, these two sets of reactions would be run out on
an agarose gel, and the band pattern will tell you if the individual is homozygous T,
homozygous C, or heterozygous T/C. This methodology has several applications,
such as amplifying certain haplotypes (when certain alleles at 2 or more SNPs occur
together on the same chromosome Linkage Disequilibrium) or detection of
recombinant chromosomes and the study of meiotic recombination.
Comparison of gene expression
Researchers have used traditional PCR as a way to estimate changes in the amount of
a gene's expression. Ribonucleic acid (RNA) is the molecule into which DNA is
transcribed prior to making a protein, and those strands of RNA that hold the
instructions for protein sequence are known as messenger RNA (mRNA). Once RNA
is isolated it can be reverse transcribed back into DNA (complementary DNA or
cDNA), at which point DNA-based PCR can be applied to amplify the gene, a method
called RT-PCR. The proportion of mRNA transcripts from a given gene in a sample
determines the relative proportion of cDNA amplified by PCR from this sample.
When cDNA products of the PCR are fractionated on an agarose gel (see Figure 3
above) a band, corresponding to a highly expressed gene, often has higher intensity,
due to its containing greater amounts of amplified DNA. This qualitative approach
may be suited for a quick analysis of gene expression in differently treated organisms
or tissues to identify levels of expression of a gene of interest. A more quantitative
RT-PCR method has been developed, called Real-time PCR.
] AIDS testing
PCR is also used in a test for HIV infection, which has been used for several years by
AIMHCF lab, which provides STD-testing services to erotic actors.
History
Polymerase chain reaction was invented by Kary Mullis [9][10]. He was awarded the
Nobel Prize in Chemistry in 1993 for his invention, only seven years after he and his
colleagues at Cetus first reduced his proposal to practice.
At the time he thought up PCR in 1983, Mullis was working in Emeryville, California
for Cetus Corporation, one of the first biotechnology companies. There, he was
charged with making short chains of DNA for other scientists. Mullis has written that
he conceived of PCR while cruising along the Pacific Coast Highway one night in his
car[9]. He was playing in his mind with a new way of analyzing changes (mutations) in
DNA when he realized that he had instead invented a method of amplifying any DNA
region through repeated cycles of duplication driven by an enzyme called DNA
polymerase.
In Scientific American, Mullis summarized the accomplishment: "Beginning with a
single molecule of the genetic material DNA, the PCR can generate 100 billion
similar molecules in an afternoon. The reaction is easy to execute. It requires no more
than a test tube, a few simple reagents, and a source of heat."[11]
DNA polymerase occurs naturally in living organisms. In cells it functions to
duplicate DNA when cells divide in mitosis and meiosis. Polymerase works by
binding to a single DNA strand and creating the complementary strand. In the first of
many original processes, 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 94°C (201°F). At this temperature, however, the DNA
polymerase used at the time were destroyed, so the enzyme had to be replenished after
the heating stage of each cycle. The original procedure was very inefficient, since it
required a great deal of time, large amounts of DNA polymerase, and continual
attention throughout the process.
Later, this original PCR process was greatly improved by the use of DNA polymerase
taken from thermophilic bacteria grown in geysers at a temperature of over 110°C
(230°F). The DNA polymerase taken from these organisms is stable at high
temperatures and, when used in PCR, does not break down when the mixture was
heated to separate the DNA strands. Since there was no longer a need to add new
DNA polymerase for each cycle, the process of copying a given DNA strand could be
simplified and automated.
One of the first thermostable DNA polymerases was obtained from Thermus
aquaticus and was called "Taq." Taq polymerase is widely used in current PCR
practice. A disadvantage of Taq is that it sometimes makes mistakes when copying
DNA, leading to mutations (errors) in the DNA sequence, since it lacks 3'→5'
proofreading exonuclease activity. Polymerases such as Pwo or Pfu, obtained from
Archaea, have proofreading mechanisms (mechanisms that check for errors) and can
significantly reduce the number of mutations that occur in the copied DNA sequence.
However these enzymes polymerise DNA at a much slower rate than Taq.
Combinations of both Taq and Pfu are available nowadays that provide both high
processivity (fast polymerisation) and high fidelity (accurate duplication of DNA).
PCR has been performed on DNA larger than 10 kilobases, but the average PCR is
only several hundred to a few thousand bases of DNA. The problem with long PCR is
that there is a balance between accuracy and processivity of the enzyme. Usually, the
longer the fragment, the greater the probability of errors.
[Patent wars
The PCR technique was patented by Cetus Corporation, where Mullis worked when
he invented the technique in 1983. The Taq polymerase enzyme was also covered by
patents. There have been several high-profile lawsuits related to the technique,
including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company
Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds
those that are still protected.
A related patent battle over the Taq polymerase enzyme is still ongoing in several
jurisdictions around the world between Roche and Promega. Interestingly, it seems
possible that the legal arguments will extend beyond the life of the original PCR and
Taq polymerase patents, which expired on March 28, 2005.[12]
References
1. ^ The history of PCR: Smithsonian Institution
Archives, Institutional History Division. Retrieved
24 June 2006.
2. ^ Cheng S, Fockler C, Barnes WM, Higuchi R
(1994). "Effective amplification of long targets from
cloned inserts and human genomic DNA". Proc Natl
Acad Sci. 91: 5695-5699. PMID 8202550.
3. ^ a b Joseph Sambrook and David W. Russel (2001).
Molecular Cloning: A Laboratory Manual, 3rd ed.,
Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press. ISBN 0-87969-576-5. Chapter 8:
In vitro Amplification of DNA by the Polymerase
Chain Reaction
4. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev
AI (2004). "Recent developments in the
optimization of thermostable DNA polymerases for
efficient applications". Trends Biotechnol. 22: 253260. PMID 15109812.
5. ^ a b D.J. Sharkey, E.R. Scalice, K.G. Christy Jr.,
S.M. Atwood, and J.L. Daiss (1994). "Antibodies as
Thermolabile Switches: High Temperature
Triggering for the Polymerase Chain Reaction".
Bio/Technology 12: 506-509.
6. ^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and
W. Bloch (1992). "Prevention of pre-PCR mispriming and primer dimerization improves lowcopy-number amplifications". Nucleic Acids
Research 20: 1717-1723.
7. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev
AI (2006). "Thermostable DNA Polymerases for a
Wide Spectrum of Applications: Comparison of a
Robust Hybrid TopoTaq to other enzymes", in
Kieleczawa J: DNA Sequencing II: Optimizing
Preparation and Cleanup. Jones and Bartlett, pp.
241-257. ISBN 0-7637338-3-0.
8. ^
http://photoscience.la.asu.edu/photosyn/courses/BIO
_343/lecture/DNAtech.html
9. ^ a b Mullis, Kary (1998). Dancing Naked in the
Mind Field. New York: Pantheon Books. ISBN 0679-44255-3.
10. ^ Rabinow, Paul (1996). Making PCR: A Story of
Biotechnology. Chicago: University of Chicago
Press. ISBN 0-226-70146-8.
11. ^ Mullis KB. The unusual origin of the polymerase
chain reaction. Sci Am 1990;262(4):56-61, 64-5.
12. ^ Advice on How to Survive the Taq Wars ¶2: GEN
Genetic Engineering News Biobusiness Channel:
Article. May 1 2006 (Vol. 26, No. 9).