Polymerase chain reaction

Polymerase chain reaction
(PCR)
Key points:
 Polymerase chain reaction, or PCR, is a technique
to make many copies of a specific DNA region in
vitro (in a test tube rather than an organism).
 PCR relies on a thermostable DNA
polymerase, Taq polymerase, and requires
DNA primers designed specifically for the DNA region
of interest.
 In PCR, the reaction is repeatedly cycled through a
series of temperature changes, which allow many
copies of the target region to be produced.
 PCR has many research and practical applications. It is
routinely used in DNA cloning, medical diagnostics,
and forensic analysis of DNA.
What is PCR?
Polymerase chain reaction (PCR) is a common
laboratory technique used to make many copies (millions
or billions!) of a particular region of DNA. This DNA region
can be anything the experimenter is interested in. For
example, it might be a gene whose function a researcher
wants to understand, or a genetic marker used by forensic
scientists to match crime scene DNA with suspects.
Typically, the goal of PCR is to make enough of the target
DNA region that it can be analyzed or used in some other
way. For instance, DNA amplified by PCR may be sent
for sequencing, visualized by gel electrophoresis,
or cloned into a plasmid for further experiments.
PCR is used in many areas of biology and medicine,
including molecular biology research, medical diagnostics,
and even some branches of ecology.
Taq polymerase
Like DNA replication in an organism, PCR requires a DNA
polymerase enzyme that makes new strands of DNA, using
existing strands as templates. The DNA polymerase
typically used in PCR is called Taq polymerase, after the
heat-tolerant bacterium from which it was isolated
(Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its
DNA polymerase is very heat-stable and is most active
around \[70 °\text C\] (a temperature at which a human
or E. coli DNA polymerase would be nonfunctional). This
heat-stability makes Taq polymerase ideal for PCR. As we'll
see, high temperature is used repeatedly in PCR
to denature the template DNA, or separate its strands.
PCR primers
Like other DNA polymerases, Taq polymerase can only
make DNA if it's given a primer, a short sequence of
nucleotides that provides a starting point for DNA
synthesis. In a PCR reaction, the experimenter determines
the region of DNA that will be copied, or amplified, by the
primers she or he chooses.
PCR primers are short pieces of single-stranded DNA,
usually around \[20\] nucleotides in length. Two primers are
used in each PCR reaction, and they are designed so that
they flank the target region (region that should be copied).
That is, they are given sequences that will make them bind
to opposite strands of the template DNA, just at the edges
of the region to be copied. The primers bind to the
template by complementary base pairing.
When the primers are bound to the template, they can be
extended by the polymerase, and the region that lies
between them will get copied.
More detailed diagram showing DNA and primer

directionality
The steps of PCR
The key ingredients of a PCR reaction are Taq polymerase,
primers, template DNA, and nucleotides (DNA building
blocks). The ingredients are assembled in a tube, along
with cofactors needed by the enzyme, and are put through
repeated cycles of heating and cooling that allow DNA to
be synthesized.
The basic steps are:
1. Denaturation (\[96 °\text C\]): Heat the reaction
strongly to separate, or denature, the DNA strands.
This provides single-stranded template for the next
step.
2. Annealing (\[55\] \[\mbox{-}\] \[65\]\[°\text C\]): Cool
the reaction so the primers can bind to their
complementary sequences on the single-stranded
template DNA.
3. Extension (\[72 °\text C\]): Raise the reaction
temperatures so Taq polymerase extends the primers,
synthesizing new strands of DNA.
This cycle repeats \[25\] \[\mbox{-}\] \[35\] times in a
typical PCR reaction, which generally takes \[2\] \[\
mbox{-}\] \[4\] hours, depending on the length of the DNA
region being copied. If the reaction is efficient (works well),
the target region can go from just one or a few copies to
billions.
That’s because it’s not just the original DNA that’s used as
a template each time. Instead, the new DNA that’s made in
one round can serve as a template in the next round of
DNA synthesis. There are many copies of the primers and
many molecules of Taq polymerase floating around in the
reaction, so the number of DNA molecules can roughly
double in each round of cycling. This pattern of exponential
growth is shown in the image below.
Using gel electrophoresis to visualize

the results of PCR
The results of a PCR reaction are usually visualized (made
visible) using gel electrophoresis. Gel electrophoresis is a
technique in which fragments of DNA are pulled through a
gel matrix by an electric current, and it separates DNA
fragments according to size. A standard, or DNA ladder, is
typically included so that the size of the fragments in the
PCR sample can be determined.
DNA fragments of the same length form a "band" on the
gel, which can be seen by eye if the gel is stained with a
DNA-binding dye. For example, a PCR reaction producing
a \[400\] base pair (bp) fragment would look like this on a
gel:
A DNA band contains many, many copies of the target DNA
region, not just one or a few copies. Because DNA is
microscopic, lots of copies of it must be present before we
can see it by eye. This is a big part of why PCR is an
important tool: it produces enough copies of a DNA
sequence that we can see or manipulate that region of
DNA.
Applications of PCR
Using PCR, a DNA sequence can be amplified millions or
billions of times, producing enough DNA copies to be
analyzed using other techniques. For instance, the DNA
may be visualized by gel electrophoresis, sent
for sequencing, or digested with restriction enzymes
and cloned into a plasmid.
PCR is used in many research labs, and it also has practical
applications in forensics, genetic testing, and diagnostics.
For instance, PCR is used to amplify genes associated with
genetic disorders from the DNA of patients (or from fetal
DNA, in the case of prenatal testing). PCR can also be used
to test for a bacterium or DNA virus in a patient's body: if
the pathogen is present, it may be possible to amplify
regions of its DNA from a blood or tissue sample.

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Polymerase chain reaction

More detailed diagram showing DNA and primer

Using gel electrophoresis to visualize

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