The polymerase
chain reaction (PCR)
is a biochemical technology inmolecular
biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies a particular DNA sequence.
Developed in
1983 by Kary Mullis, PCR is now a common and often
indispensable technique used in medical and biological research labs for a
variety of applications.These include DNA cloning for sequencing,
DNA-based phylogeny, or functional
analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic
sciencesand paternity
testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was
awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.
The method
relies on thermal cycling,
consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short
DNA fragments) containing sequences complementary to the target region along
with a DNA polymerase (after which the method is named) are
key components to 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 DNA template is exponentially amplified. PCR can be extensively
modified to perform a wide array of genetic manipulations.
Almost all
PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase,
an enzyme originally isolated from the bacterium Thermus
aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA
building-blocks, thenucleotides, by using single-stranded DNA as a template and DNA
oligonucleotides (also
called DNA primers), which are required for
initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling,
i.e., alternately heating and cooling the PCR sample to a defined series of
temperature steps. These thermal cycling steps are necessary first to
physically separate the two strands in a DNA double helix at a high temperature
in a process called DNA melting.
At a lower temperature, each strand is then used as thetemplate in DNA synthesis by the DNA polymerase
to selectively amplify the target DNA. The selectivity of PCR results from the
use ofprimers that
are complementary to the DNA region targeted for
amplification under specific thermal cycling conditions.
Typically, PCR consists of a series of 20-40
repeated temperature changes, called cycles, with each cycle commonly
consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The
cycling is often preceded by a single temperature step (called hold) at a high temperature
(>90°C), 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. These include the enzyme used for
DNA synthesis, the concentration of divalent ions and dNTPs in the reaction,
and the melting temperature (Tm) of the primers.
§
Initialization step: This step consists of heating the reaction to
a temperature of 94–96 °C (or 98 °C if extremely thermostable
polymerases are used), which is held for 1–9 minutes. It is only required for
DNA polymerases that require heat activation by hot-start PCR.
§
Denaturation step: This step is the first
regular cycling event and consists of heating the reaction to 94–98 °C for
20–30 seconds. It causes DNA melting of the DNA template by disrupting the
hydrogen bonds between complementary bases, yielding single-stranded DNA
molecules.
§
Annealing step: The reaction
temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of
the primers to the single-stranded DNA template. Typically the annealing
temperature is about 3-5 degrees Celsius below the Tm of the primers used.
Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very
closely matches the template sequence. The polymerase binds to the
primer-template hybrid and begins DNA formation.
§
Extension/elongation step: The temperature at this step depends on
the DNA polymerase used; Taq polymerase has its optimum activitytemperature
at 75–80 °C,and
commonly a temperature of 72 °C is used with this enzyme. At this step the
DNA polymerase synthesizes a new DNA strand complementary to the DNA template
strand by adding dNTPs that are complementary to the template in 5' to 3'
direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl groupat the end of the nascent
(extending) DNA strand. 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
per minute. Under optimum conditions, i.e., if there are no limitations due to
limiting substrates or reagents, at each extension step, the amount of DNA
target is doubled, leading to exponential (geometric) amplification of the
specific DNA fragment.
§
Final elongation: This single step is occasionally performed at
a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully extended.
§
Final hold: This step at 4–15 °C for an indefinite
time may be employed for short-term storage of the reaction.
To check whether the PCR generated the anticipated DNA
fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is 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 contains DNA
fragments of known size, run on the gel alongside the PCR products 
A thermal cycler for PCR
An older model three-temperature thermal cycler for PCR
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 blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
Placing a strip of eight PCR tubes, each containing a 100 μl reaction mixture, into the PCR machine
A strip of eight PCR tubes, each containing a 100 μl
reaction mixture
Application of PCR
Main article: Applications of PCR
[edit]Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, 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 recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E. coli) can be rapidly screened by PCR for correct DNA vector constructs.[18] 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.[citation needed]
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.[citation needed]
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