POLYMERASE CHAIN REACTION (PCR) is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of DNA
across several orders of magnitude, generating thousands to millions
of copies of a particular
DNA sequence . It is an easy, cheap, and
reliable way to repeatedly replicate a focused segment of DNA, a
concept which is applicable to numerous fields in modern biology and
Developed in 1983 by
Kary Mullis , PCR is now a common and often
indispensable technique used in clinical and research laboratories for
a broad variety of applications. These include
DNA cloning for
sequencing , gene cloning and manipulation, gene mutagenesis;
construction of DNA-based phylogenies , or functional analysis of
genes ; diagnosis and monitoring of hereditary diseases ;
amplification of ancient DNA; analysis of genetic fingerprints for
DNA profiling (for example, in forensic science and parentage testing
); and detection of pathogens in nucleic acid tests for the diagnosis
of infectious diseases . In 1993, Mullis was awarded the Nobel Prize
in Chemistry along with Michael Smith for his work on PCR.
Placing a strip of eight PCR tubes into a thermal cycler
The vast majority of PCR methods rely on thermal cycling , which
involves exposing the reactants to cycles of repeated heating and
cooling, permitting different temperature-dependent
DNA melting and enzyme -driven DNA
replication—to quickly proceed many times in sequence. Primers
DNA fragments) containing sequences complementary to the target
region, along with a
DNA polymerase , after which the method is named,
enable selective and repeated amplification. As PCR progresses, the
DNA generated is itself used as a template for replication, setting in
motion a chain reaction in which the original
DNA template is
exponentially amplified. The simplicity of the basic principle
underlying PCR means it can be extensively modified to perform a wide
array of genetic manipulations . PCR is not generally considered to be
DNA method, as it does not involve cutting and pasting
DNA, only amplification of existing sequences.
Almost all PCR applications employ a heat-stable
DNA polymerase, such
Taq polymerase , an enzyme originally isolated from the
Thermus aquaticus . This
enzymatically assembles a new
DNA strand from free nucleotides , the
building blocks of DNA, by using single-stranded
DNA as a template and
DNA oligonucleotides (the primers mentioned above) to initiate DNA
In the first step, the two strands of the
DNA double helix are
physically separated at a high temperature in a process called DNA
melting . In the second step, the temperature is lowered and the two
DNA strands become templates for
DNA polymerase to selectively amplify
the target DNA. The selectivity of PCR results from the use of primers
that are complementary to the
DNA region targeted for amplification
under specific thermal cycling conditions.
* 1 Principles
* 1.1 Procedure
* 1.2 Stages
* 2 Optimization
* 3 Applications
* 3.1 Selective
* 3.2 Amplification and quantification of
* 3.3 Medical applications
Infectious disease applications
* 3.5 Forensic applications
* 3.6 Research applications
* 4 Advantages
* 5 Limitations
* 6 Variations
* 7 History
* 7.1 Patent disputes
* 8 See also
* 9 References
* 10 External links
A thermal cycler for PCR An older model three-temperature
thermal cycler for PCR
PCR amplifies a specific region of a
DNA strand (the
Most PCR methods amplify
DNA fragments of between 0.1 and 10 kilo base
pairs (kbp), although some techniques allow for amplification of
fragments up to 40 kbp in size. The amount of amplified product is
determined by the available substrates in the reaction, which become
limiting as the reaction progresses.
A basic PCR set-up requires several components and reagents,
DNA template that contains the
DNA target region to amplify
DNA polymerase , an enzyme that polymerizes new
Taq polymerase is especially common, as it is more
likely to remain intact during the high-temperature
DNA primers that are complementary to the 3\' (three prime)
ends of each of the sense and anti-sense strands of the
DNA polymerase can only bind to and elongate from a double-stranded
region of DNA; without primers there is no double-stranded initiation
site at which the polymerase can bind); specific primers that are
complementary to the
DNA target region are selected beforehand, and
are often custom-made in a laboratory or purchased from commercial
* deoxynucleoside triphosphates, or dNTPs (sometimes called
"deoxynucleotide triphosphates"; nucleotides containing triphosphate
groups), the building blocks from which the
DNA polymerase synthesizes
* a buffer solution providing a suitable chemical environment for
optimum activity and stability of the
* bivalent cations , typically magnesium (Mg) or manganese (Mn)
ions; Mg2+ is the most common, but Mn2+ can be used for PCR-mediated
DNA mutagenesis , as a higher Mn2+ concentration increases the error
* monovalent cations, typically potassium (K) ions
The reaction is commonly carried out in a volume of 10–200 μl in
small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler . The
thermal cycler heats and cools the reaction tubes to achieve the
temperatures required at each step of the reaction (see below). Many
modern thermal cyclers make use of the Peltier effect , which permits
both heating and cooling of the block holding the PCR tubes simply by
reversing the electric current. Thin-walled reaction tubes permit
favorable thermal conductivity to allow for rapid thermal
equilibration. Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube. Older thermal cyclers
lacking a heated lid require a layer of oil on top of the reaction
mixture or a ball of wax inside the tube.
Typically, PCR consists of a series of 20–40 repeated temperature
changes, called cycles, with each cycle commonly consisting of two or
three discrete temperature steps (see figure below). The cycling is
often preceded by a single temperature step at a very high temperature
(>90 °C (194 °F)), and followed by one hold at the end for final
product extension or brief storage. The temperatures used and the
length of time they are applied in each cycle depend on a variety of
parameters, including the enzyme used for
DNA synthesis, the
concentration of bivalent ions and dNTPs in the reaction, and the
melting temperature (Tm) of the primers. The individual steps common
to most PCR methods are as follows:
* INITIALIZATION: This step is only required for
that require heat activation by hot-start PCR . It consists of
heating the reaction chamber to a temperature of 94–96 °C
(201–205 °F), or 98 °C (208 °F) if extremely thermostable
polymerases are used, which is then held for 1–10 minutes.
* DENATURATION : This step is the first regular cycling event and
consists of heating the reaction chamber to 94–98 °C (201–208
°F) for 20–30 seconds. This causes
DNA melting , or denaturation,
of the double-stranded
DNA template by breaking the hydrogen bonds
between complementary bases, yielding two single-stranded DNA
* ANNEALING : In the next step, the reaction temperature is lowered
to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing
of the primers to each of the single-stranded
DNA templates. Two
different primers are typically included in the reaction mixture: one
for each of the two single-stranded complements containing the target
region. The primers are single-stranded sequences themselves, but are
much shorter than the length of the target region, complementing only
very short sequences at the 3' end of each strand.
It is critical to determine a proper temperature for the annealing
step because efficiency and specificity are strongly affected by the
annealing temperature. This temperature must be low enough to allow
for hybridization of the primer to the strand, but high enough for the
hybridization to be specific, i.e., the primer should bind only to a
perfectly complementary part of the strand, and nowhere else. If the
temperature is too low, the primer may bind imperfectly. If it is too
high, the primer may not bind at all. A typical annealing temperature
is about 3–5 °C below the Tm of the primers used. Stable hydrogen
bonds between complementary bases are formed only when the primer
sequence very closely matches the template sequence. During this step,
the polymerase binds to the primer-template hybrid and begins DNA
* EXTENSION/ELONGATION: The temperature at this step depends on the
DNA polymerase used; the optimum activity temperature for Taq
polymerase is approximately 75–80 °C (167–176 °F), though a
temperature of 72 °C (162 °F) is commonly used with this enzyme. In
this step, the
DNA polymerase synthesizes a new
complementary to the
DNA template strand by adding free dNTPs from the
reaction mixture that are complementary to the template in the
5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs
with the 3'-hydroxy group at the end of the nascent (elongating) DNA
strand. The precise time required for elongation depends both on the
DNA polymerase used and on the length of the
DNA target region to
amplify. As a rule of thumb, at their optimal temperature, most DNA
polymerases polymerize a thousand bases per minute. Under optimal
conditions (i.e., if there are no limitations due to limiting
substrates or reagents), at each extension/elongation step, the number
DNA target sequences is doubled. With each successive cycle, the
original template strands plus all newly generated strands become
template strands for the next round of elongation, leading to
exponential (geometric) amplification of the specific
The processes of denaturation, annealing and elongation constitute a
single cycle. Multiple cycles are required to amplify the
to millions of copies. The formula used to calculate the number of DNA
copies formed after a given number of cycles is 2n, where n is the
number of cycles. Thus, a reaction set for 30 cycles results in 230,
or 1073741824, copies of the original double-stranded
* FINAL ELONGATION: This single step is optional, but is performed
at a temperature of 70–74 °C (158–165 °F) (the temperature range
required for optimal activity of most polymerases used in PCR) for
5–15 minutes after the last PCR cycle to ensure that any remaining
DNA is fully elongated.
* FINAL HOLD: The final step cools the reaction chamber to 4–15
°C (39–59 °F) for an indefinite time, and may be employed for
short-term storage of the PCR products.
Ethidium bromide -stained PCR products after gel
electrophoresis . Two sets of primers were used to amplify a target
sequence from three different tissue samples. No amplification is
present in sample #1;
DNA bands in sample #2 and #3 indicate
successful amplification of the target sequence. The gel also shows a
positive control, and a
DNA ladder containing
DNA fragments of defined
length for sizing the bands in the experimental PCRs.
To check whether the PCR successfully generated the anticipated DNA
target region (also sometimes referred to as the amplimer or amplicon
), agarose gel electrophoresis may be employed for size separation of
the PCR products. The size(s) of PCR products is determined by
comparison with a
DNA ladder , a molecular weight marker which
DNA fragments of known size run on the gel alongside the PCR
As with other chemical reactions, the reaction rate and efficiency of
PCR are affected by limiting factors. Thus, the entire PCR process can
further be divided into three stages based on reaction progress:
* Exponential amplification: At every cycle, the amount of product
is doubled (assuming 100% reaction efficiency). The reaction is very
sensitive: only minute quantities of
DNA must be present.
* Leveling off stage: The reaction slows as the
DNA polymerase loses
activity and as consumption of reagents such as dNTPs and primers
causes them to become limiting.
* Plateau: No more product accumulates due to exhaustion of reagents
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
DNA is addressed with lab protocols and procedures that
separate pre-PCR mixtures from potential
DNA contaminants. This
usually involves spatial separation of PCR-setup areas from areas for
analysis or purification of PCR products, use of disposable
plasticware, 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. Addition of reagents, such as formamide , in buffer systems may
increase the specificity and yield of PCR. Computer simulations of
theoretical PCR results (Electronic PCR ) may be performed to assist
in primer design.
PCR allows isolation of
DNA fragments from genomic
DNA by selective
amplification of a specific region of DNA. This use of PCR augments
many ways, such as generating hybridization probes for Southern or
northern hybridization and
DNA cloning , which require larger amounts
of DNA, representing a specific
DNA region. PCR supplies these
techniques with high amounts of pure DNA, enabling analysis of DNA
samples even from very small amounts of starting material.
Other applications of PCR include
DNA sequencing to determine unknown
PCR-amplified sequences in which one of the amplification primers may
be used in Sanger sequencing, isolation of a
DNA sequence to expedite
DNA technologies involving the insertion of a
into a plasmid , phage , or cosmid (depending on size) or the genetic
material of another organism. Bacterial colonies (such as E. coli )
can be rapidly screened by PCR for correct
DNA vector constructs. PCR
may also be used for genetic fingerprinting ; a forensic technique
used to identify a person or organism by comparing experimental DNAs
through different PCR-based methods.
Some PCR 'fingerprints' methods have high discriminative power and
can be used to identify genetic relationships between individuals,
such as parent-child or between siblings, and are used in paternity
testing (Fig. 4). This technique may also be used to determine
evolutionary relationships among organisms when certain molecular
clocks are used (i.e., the
16S rRNA and recA genes of microorganisms).
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
AMPLIFICATION AND QUANTIFICATION OF DNA
See also: Use of
DNA in forensic entomology
Because PCR amplifies the regions of
DNA that it targets, PCR can be
used to analyze extremely small amounts of sample. This is often
critical for forensic analysis , when only a trace amount of
available as evidence. PCR may also be used in the analysis of ancient
DNA that is tens of thousands of years old. These PCR-based 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 and the body of English king Richard
Quantitative PCR methods allow the estimation of the amount of a
given sequence present in a sample—a technique often applied to
quantitatively determine levels of gene expression . Quantitative PCR
is an established tool for
DNA quantification that measures the
DNA product after each round of PCR amplification.
qPCR allows the quantification and detection of a specific DNA
sequence in real time since it measures concentration while the
synthesis process is taking place. There are two methods for
simultaneous detection and quantification. The first method is
fluorescent dyes that are retained nonspecifically in between the
double strands. The second method are probes that code for specific
sequences and are fluorescently labeled. Detection of
DNA using these
methods can only be seen after the hybridization of probes with its
DNA takes place. An interesting technique combination is
real-time PCR and reverse transcription (RT-qPCR). This sophisticated
technique allows for the quantification of a small quantity of RNA.
Through this combined technique, m
RNA is converted to cDNA, which is
further quantified using qPCR. This technique lowers the possibility
of error at the end point of PCR, increasing chances for detection of
genes associated with genetic diseases such as cancer. Laboratories
use RT-qPCR for the purpose of measuring gene regulation sensitively.
PCR has been applied to a large number of medical procedures:
* The first application of PCR was for GENETIC TESTING , where a
DNA is analyzed for the presence of genetic disease
mutations . Prospective parents can be tested for being genetic
carriers , or their children might be tested for actually being
affected by a disease .
DNA samples for prenatal testing can be
obtained by amniocentesis , chorionic villus sampling , or even by the
analysis of rare fetal cells circulating in the mother's bloodstream.
PCR analysis is also essential to preimplantation genetic diagnosis ,
where individual cells of a developing embryo are tested for
* PCR can also be used as part of a sensitive test for TISSUE TYPING
, vital to organ transplantation . As of 2008, there is even a
proposal to replace the traditional antibody-based tests for blood
type with PCR-based tests.
* Many forms of cancer involve alterations to ONCOGENES . By using
PCR-based tests to study these mutations, therapy regimens can
sometimes be individually customized to a patient. PCR permits early
diagnosis of malignant diseases such as leukemia and lymphomas , which
is currently the highest-developed in cancer research and is already
being used routinely. PCR assays can be performed directly on genomic
DNA samples to detect translocation-specific malignant cells at a
sensitivity that is at least 10,000 fold higher than that of other
methods. PCR is very useful in the medical field since it allows for
the isolation and amplification of tumor suppressors. Quantitative PCR
for example, can be used to quantify and analyze single cells, as well
as recognize DNA, m
RNA and protein confirmations and combinations.
INFECTIOUS DISEASE APPLICATIONS
PCR allows for rapid and highly specific diagnosis of infectious
diseases, including those caused by bacteria or viruses. PCR also
permits identification of non-cultivatable or slow-growing
microorganisms such as mycobacteria , anaerobic bacteria , or viruses
from tissue culture assays and animal models . The basis for PCR
diagnostic applications in microbiology is the detection of infectious
agents and the discrimination of non-pathogenic from pathogenic
strains by virtue of specific genes.
Characterization and detection of infectious disease organisms have
been revolutionized by PCR in the following ways:
* The HUMAN IMMUNODEFICIENCY VIRUS (or
HIV ), is a difficult target
to find and eradicate. The earliest tests for infection relied on the
presence of antibodies to the virus circulating in the bloodstream.
However, antibodies don't appear until many weeks after infection,
maternal antibodies mask the infection of a newborn, and therapeutic
agents to fight the infection don't affect the antibodies. PCR tests
have been developed that can detect as little as one viral genome
DNA of over 50,000 host cells. Infections can be detected
earlier, donated blood can be screened directly for the virus,
newborns can be immediately tested for infection, and the effects of
antiviral treatments can be quantified .
* Some disease organisms, such as that for TUBERCULOSIS , are
difficult to sample from patients and slow to be grown in the
laboratory. PCR-based tests have allowed detection of small numbers of
disease organisms (both live or dead), in convenient samples .
Detailed genetic analysis can also be used to detect antibiotic
resistance, allowing immediate and effective therapy. The effects of
therapy can also be immediately evaluated.
* The spread of a DISEASE ORGANISM through populations of domestic
or wild animals can be monitored by PCR testing. In many cases, the
appearance of new virulent sub-types can be detected and monitored.
The sub-types of an organism that were responsible for earlier
epidemics can also be determined by PCR analysis.
DNA can be detected by PCR. The primers used must be
specific to the targeted sequences in the
DNA of a virus, and PCR can
be used for diagnostic analyses or
DNA sequencing of the viral genome.
The high sensitivity of PCR permits virus detection soon after
infection and even before the onset of disease. Such early detection
may give physicians a significant lead time in treatment. The amount
of virus ("viral load ") in a patient can also be quantified by
DNA quantitation techniques (see below).
The development of PCR-based genetic (or
DNA ) fingerprinting
protocols has seen widespread application in forensics :
* In its most discriminating form, GENETIC FINGERPRINTING can
uniquely discriminate any one person from the entire population of the
world . Minute samples of
DNA can be isolated from a crime scene , and
compared to that from suspects, or from a
DNA database of earlier
evidence or convicts. Simpler versions of these tests are often used
to rapidly rule out suspects during a criminal investigation. Evidence
from decades-old crimes can be tested, confirming or exonerating the
people originally convicted.
DNA typing has been an effective way of identifying or
exonerating criminal suspects due to analysis of evidence discovered
at a crime scene. The human genome has many repetitive regions that
can be found within gene sequences or in non-coding regions of the
genome. Specifically, up to 40% of human
DNA is repetitive. There are
two distinct categories for these repetitive, non-coding regions in
the genome. The first category is called variable number tandem
repeats (VNTR), which are 10-100 base pairs long and the second
category is called short tandem repeats (STR) and these consist of
repeated 2-10 base pair sections. PCR is used to amplify several
well-known VNTRs and STRs using primers that flank each of the
repetitive regions. The sizes of the fragments obtained from any
individual for each of the STRs will indicate which alleles are
present. By analyzing several STRs for an individual, a set of alleles
for each person will be found that statistically is likely to be
unique. Researchers have identified the complete sequence of the
human genome. This sequence can be easily accessed through the NCBI
website and is used in many real-life applications. For example, the
FBI has compiled a set of
DNA marker sites used for identification,
and these are called the Combined
DNA Index System (CODIS) DNA
database. Using this database enables statistical analysis to be used
to determine the probability that a
DNA sample will match. PCR is a
very powerful and significant analytical tool to use for forensic DNA
typing because researchers only need a very small amount of the target
DNA to be used for analysis. For example, a single human hair with
attached hair follicle has enough
DNA to conduct the analysis.
Similarly, a few sperm, skin samples from under the fingernails, or a
small amount of blood can provide enough
DNA for conclusive analysis.
* Less discriminating forms of
DNA fingerprinting can help in DNA
PATERNITY TESTING , where an individual is matched with their close
DNA from unidentified human remains can be tested, and
compared with that from possible parents, siblings, or children.
Similar testing can be used to confirm the biological parents of an
adopted (or kidnapped) child. The actual biological father of a
newborn can also be confirmed (or ruled out).
PCR has been applied to many areas of research in molecular genetics:
* PCR allows rapid production of short pieces of DNA, even when not
more than the sequence of the two primers is known. This ability of
PCR augments many methods, such as generating HYBRIDIZATION PROBES for
Southern or northern blot hybridization. PCR supplies these techniques
with large amounts of pure DNA, sometimes as a single strand, enabling
analysis even from very small amounts of starting material.
* The task of
DNA SEQUENCING can also be assisted by PCR. Known
DNA can easily be produced from a patient with a genetic
disease mutation. Modifications to the amplification technique can
extract segments from a completely unknown genome, or can generate
just a single strand of an area of interest.
* PCR has numerous applications to the more traditional process of
DNA CLONING . It can extract segments for insertion into a vector from
a larger genome, which may be only available in small quantities.
Using a single set of 'vector primers', it can also analyze or extract
fragments that have already been inserted into vectors. Some
alterations to the PCR protocol can GENERATE MUTATIONS (general or
site-directed) of an inserted fragment.
* SEQUENCE-TAGGED SITES is a process where PCR is used as an
indicator that a particular segment of a genome is present in a
particular clone. The
Human Genome Project
Human Genome Project found this application
vital to mapping the cosmid clones they were sequencing, and to
coordinating the results from different laboratories.
* An exciting application of PCR is the phylogenic analysis of DNA
from ANCIENT SOURCES , such as that found in the recovered bones of
Neanderthals , or from frozen tissues of mammoths . In some cases the
DNA from these sources might be reassembled during the
early stages of amplification.
* A common application of PCR is the study of patterns of GENE
EXPRESSION . Tissues (or even individual cells) can be analyzed at
different stages to see which genes have become active, or which have
been switched off. This application can also use quantitative PCR to
quantitate the actual levels of expression
* The ability of PCR to simultaneously amplify several loci from
individual sperm has greatly enhanced the more traditional task of
GENETIC MAPPING by studying chromosomal crossovers after meiosis .
Rare crossover events between very close loci have been directly
observed by analyzing thousands of individual sperms. Similarly,
unusual deletions, insertions, translocations, or inversions can be
analyzed, all without having to wait (or pay) for the long and
laborious processes of fertilization, embryogenesis, etc.
PCR has a number of advantages. It is fairly simple to understand and
to use, and produces results rapidly. The technique is highly
sensitive with the potential to produce millions to billions of copies
of a specific product for sequencing, cloning, and analysis. qRT-PCR
shares the same advantages as the PCR, with an added advantage of
quantification of the synthesized product. Therefore, it has its uses
to analyze alterations of gene expression levels in tumors, microbes,
or other disease states.
One major limitation of PCR is that prior information about the
target sequence is necessary in order to generate the primers that
will allow its selective amplification. This means that, typically,
PCR users must know the precise sequence(s) upstream of the target
region on each of the two single-stranded templates in order to ensure
DNA polymerase properly binds to the primer-template hybrids
and subsequently generates the entire target region during DNA
Like all enzymes,
DNA polymerases are also prone to error, which in
turn causes mutations in the PCR fragments that are generated.
Variants of PCR
* Allele-specific PCR : a diagnostic or cloning technique based on
single-nucleotide variations (SNVs not to be confused with SNPs )
(single-base differences in a patient). It requires prior knowledge of
DNA sequence, including differences between alleles , and uses
primers whose 3' ends encompass the SNV (base pair buffer around SNV
usually incorporated). PCR amplification under stringent conditions is
much less efficient in the presence of a mismatch between template and
primer, so successful amplification with an SNP-specific primer
signals presence of the specific SNP in a sequence. See SNP
genotyping for more information.
* Assembly PCR or Polymerase Cycling Assembly (PCA): artificial
synthesis of long
DNA sequences 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 determine the order of the PCR fragments, thereby selectively
producing the final long
Asymmetric PCR : preferentially amplifies one
DNA strand in a
DNA template. It is used in sequencing and
hybridization probing where amplification of only one of the two
complementary strands is required. PCR is carried out as usual, but
with a great excess of the primer for the strand targeted for
amplification. Because of 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 Linear-After-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
* Convective PCR : a pseudo-isothermal way of performing PCR.
Instead of repeatedly heating and cooling the PCR mixture, the
solution is subjected to a thermal gradient. The resulting thermal
instability driven convective flow automatically shuffles the PCR
reagents from the hot and cold regions repeatedly enabling PCR.
Parameters such as thermal boundary conditions and geometry of the PCR
enclosure can be optimized to yield robust and rapid PCR by harnessing
the emergence of chaotic flow fields. Such convective flow PCR setup
significantly reduces device power requirement and operation time.
* Dial-out PCR : a highly parallel method for retrieving accurate
DNA molecules for gene synthesis. A complex library of
is modified with unique flanking tags before massively parallel
sequencing. Tag-directed primers then enable the retrieval of
molecules with desired sequences by PCR.
Digital PCR (dPCR): used to measure the quantity of a target DNA
sequence in a
DNA sample. The
DNA sample is highly diluted so that
after running many PCRs in parallel, some of them do not receive a
single molecule of the target DNA. The target
DNA concentration is
calculated using the proportion of negative outcomes. Hence the name
Helicase-dependent amplification : similar to traditional PCR, but
uses a constant temperature rather than cycling through denaturation
and annealing/extension cycles.
DNA helicase , an enzyme that unwinds
DNA, is used in place of thermal denaturation.
Hot start PCR : a technique that reduces non-specific
amplification during the initial set up stages of the PCR. It may be
performed manually by heating the reaction components to the
denaturation temperature (e.g., 95 °C) before adding the polymerase.
Specialized enzyme systems have been developed that inhibit the
polymerase's activity at ambient temperature, either by the binding of
an antibody or by the presence of covalently bound inhibitors that
dissociate only 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
In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR)
refers to computational tools used to calculate theoretical polymerase
chain reaction results using a given set of primers (probes ) to
DNA sequences from a sequenced genome or transcriptome . In
silico PCR was proposed as an educational tool for molecular biology.
* Intersequence-specific PCR (ISSR): a PCR method for DNA
fingerprinting that amplifies regions between simple sequence repeats
to produce a unique fingerprint of amplified fragment lengths.
* Inverse PCR : is commonly used to identify the flanking sequences
around genomic inserts. It involves a series of
DNA digestions and
self ligation , resulting in known sequences at either end of the
* Ligation-mediated PCR : uses small
DNA linkers ligated to the DNA
of interest and multiple primers annealing to the
DNA linkers; it has
been used for
DNA sequencing , genome walking , and
DNA footprinting .
Methylation-specific PCR (MSP): developed by
Stephen Baylin and
Jim Herman at the Johns Hopkins School of Medicine, and 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 PCRs 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
* Miniprimer PCR : uses a thermostable polymerase (S-Tbr) that can
extend from short primers ("smalligos") as short as 9 or 10
nucleotides. This method permits PCR targeting to smaller primer
binding regions, and is used to amplify conserved
DNA sequences, such
as the 16S (or eukaryotic 18S) r
Multiplex ligation-dependent probe amplification (MLPA): permits
amplifying multiple targets with a single primer pair, thus avoiding
the resolution limitations of multiplex PCR (see below).
Multiplex-PCR : consists of multiple primer sets within a single
PCR mixture to produce amplicons of varying sizes that are specific to
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. That is, their base pair length should be different enough to
form distinct bands when visualized by gel electrophoresis .
* Nanoparticle-Assisted PCR (nanoPCR) : In recent years, it has been
reported that some nanoparticles (NPs) can enhance the efficiency of
PCR (thus being called nanoPCR), and some even perform better than the
original PCR enhancers. It was also found that quantum dots (QDs) can
improve PCR specificity and efficiency. Single-walled carbon nanotubes
(SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are efficient in
enhancing the amplification of long PCR. Carbon nanopowder (CNP) was
reported be able to improve the efficiency of repeated PCR and long
PCR. ZnO, TiO2, and Ag NPs were also found to increase PCR yield.
Importantly, already known data has indicated that non-metallic NPs
retained acceptable amplification fidelity. Given that many NPs are
capable of enhancing PCR efficiency, it is clear that there is likely
to be great potential for nanoPCR technology improvements and product
Nested PCR : increases the specificity of
DNA amplification, by
reducing background due to non-specific amplification of DNA. Two sets
of primers are used in two successive PCRs. 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) are then used in a second PCR with a set of
primers whose binding sites are completely or partially different from
and located 3' of each of the primers used in the first reaction.
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.
Overlap-extension PCR or Splicing by overlap extension (SOEing) :
a genetic engineering technique that is used to splice together two or
DNA fragments that contain complementary sequences. It is used to
DNA pieces containing genes, regulatory sequences, or mutations;
the technique enables creation of specific and long
DNA constructs. It
can also introduce deletions, insertions or point mutations into a DNA
* PAN-AC : uses isothermal conditions for amplification, and may be
used in living cells.
* quantitative PCR (qPCR): used to measure the quantity of a target
sequence (commonly in real-time). It quantitatively measures starting
amounts of DNA, cDNA, or RNA. quantitative PCR is commonly used to
determine whether a
DNA sequence is present in a sample and the number
of its copies in the sample.
Quantitative PCR has a very high degree
Quantitative PCR methods use fluorescent dyes, such as
Sybr Green, EvaGreen or fluorophore -containing
DNA probes, such as
TaqMan , to measure the amount of amplified product in real time. It
is also sometimes abbreviated to
RT-PCR (real-time PCR) but this
abbreviation should be used only for reverse transcription PCR . qPCR
is the appropriate contractions for quantitative PCR (real-time PCR).
* Reverse Transcription PCR (
RT-PCR ): for amplifying
DNA from RNA.
Reverse transcriptase reverse transcribes
RNA into c
DNA , which is
then amplified by PCR.
RT-PCR is widely used in expression profiling ,
to determine the expression of a gene or to identify the sequence of
RNA transcript, including transcription start and termination
sites. If the genomic
DNA sequence of a gene is known,
RT-PCR can be
used 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 RACE-PCR (Rapid Amplification of c
* Solid Phase PCR : encompasses multiple meanings, including Polony
Amplification (where PCR colonies are derived in a gel matrix, for
example), Bridge PCR (primers are covalently linked to a
solid-support surface), conventional Solid Phase PCR (where Asymmetric
PCR is applied in the presence of solid support bearing primer with
sequence matching one of the aqueous primers) and Enhanced Solid Phase
PCR (where conventional Solid Phase PCR can be improved by employing
high Tm and nested solid support primer with optional application of a
thermal 'step' to favour solid support priming).
* Suicide PCR: typically used in paleogenetics or other studies
where avoiding false positives and ensuring the specificity of the
amplified fragment is the highest priority. It was originally
described in a study to verify the presence of the microbe Yersinia
pestis in dental samples obtained from 14th Century graves of people
supposedly killed by plague during the medieval
Black Death epidemic.
The method prescribes the use of any primer combination only once in a
PCR (hence the term "suicide"), which should never have been used in
any positive control PCR reaction, and the primers should always
target a genomic region never amplified before in the lab using this
or any other set of primers. This ensures that no contaminating DNA
from previous PCR reactions is present in the lab, which could
otherwise generate false positives.
* Thermal asymmetric interlaced PCR (TAIL-PCR ): for isolation of an
unknown sequence flanking a known sequence. Within the known sequence,
TAIL-PCR uses a nested pair of primers with differing annealing
temperatures; a degenerate primer is used to amplify in the other
direction from the unknown sequence.
Touchdown PCR (Step-down PCR): 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 (3–5 °C) above the Tm
of the primers used, while at the later cycles, it is a few degrees
(3–5 °C) 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
* Universal Fast Walking : for genome walking and genetic
fingerprinting using a more specific 'two-sided' PCR than conventional
'one-sided' approaches (using only one gene-specific primer and one
general primer—which can lead to artefactual 'noise') by virtue of
a mechanism involving lariat structure formation. Streamlined
derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for
rapid amplification of genomic
DNA ends), 5'RACE LaNe and 3'RACE
Diagrammatic representation of an example primer pair. The use
of primers in an in vitro assay to allow
DNA synthesis was a major
innovation that allowed the development of PCR. Main article:
History of polymerase chain reaction
A 1971 paper in the
Journal of Molecular Biology by Kjell Kleppe
(no) and co-workers in the laboratory of H. Gobind Khorana first
described a method using an enzymatic assay to replicate a short DNA
template with primers in vitro. However, this early manifestation of
the basic PCR principle did not receive much attention at the time,
and the invention of the polymerase chain reaction in 1983 is
generally credited to
Kary Mullis . "Baby Blue", a 1986
prototype machine for doing PCR
When Mullis developed the PCR in 1983, he was working in Emeryville ,
Cetus Corporation , one of the first biotechnology
companies. There, he was responsible for synthesizing short chains of
DNA. Mullis has written that he conceived of PCR while cruising along
the Pacific Coast Highway one night in his car. 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 DNA
Scientific American , Mullis summarized the procedure:
"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." He was awarded the Nobel
Prize in Chemistry in 1993 for his invention, seven years after he
and his colleagues at Cetus first put his proposal to practice.
However, some controversies have remained about the intellectual and
practical contributions of other scientists to Mullis' work, and
whether he had been the sole inventor of the PCR principle (see
At the core of the PCR method is the use of a suitable
able to withstand the high temperatures of >90 °C (194 °F) required
for separation of the two
DNA strands in the
DNA double helix after
each replication cycle. The
DNA polymerases initially employed for in
vitro experiments presaging PCR were unable to withstand these high
temperatures. So the early procedures for
DNA replication were very
inefficient and time-consuming, and required large amounts of DNA
polymerase and continuous handling throughout the process.
The discovery in 1976 of
Taq polymerase — a
DNA polymerase purified
from the thermophilic bacterium ,
Thermus aquaticus , which naturally
lives in hot (50 to 80 °C (122 to 176 °F)) environments such as hot
springs — paved the way for dramatic improvements of the PCR method.
DNA polymerase isolated from T. aquaticus is stable at high
temperatures remaining active even after
DNA denaturation, thus
obviating the need to add new
DNA polymerase after each cycle. This
allowed an automated thermocycler-based process for
The PCR technique was patented by
Kary Mullis and assigned to Cetus
Corporation , where Mullis worked when he invented the technique in
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
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 . The legal arguments have extended beyond the lives of the
original PCR and
Taq polymerase patents, which expired on March 28,
Loop-mediated isothermal amplification
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