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What Is PCR?: T. Aquaticus Lives in Hot Springs and Hydrothermal Vents. Its DNA Polymerase Is Very Heat-Stable

PCR is a common laboratory technique used to make many copies of a particular region of DNA. It works by using the enzyme Taq polymerase and primers to denature and copy the DNA in repeated heating and cooling cycles, exponentially increasing the number of copies of the target DNA region.

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Faiz Rasool
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61 views

What Is PCR?: T. Aquaticus Lives in Hot Springs and Hydrothermal Vents. Its DNA Polymerase Is Very Heat-Stable

PCR is a common laboratory technique used to make many copies of a particular region of DNA. It works by using the enzyme Taq polymerase and primers to denature and copy the DNA in repeated heating and cooling cycles, exponentially increasing the number of copies of the target DNA region.

Uploaded by

Faiz Rasool
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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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 (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 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.
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 ℃): Heat the reaction strongly to separate, or denature, the DNA
strands. This provides single-stranded template for the next step.
2. Annealing ( 55-65 ℃ ): Cool the reaction so the primers can bind to their
complementary sequences on the single-stranded template DNA.
3. Extension ( 72 ℃): Raise the reaction temperatures so Taq polymerase extends the
primers, synthesizing new strands of DNA.
This cycle repeats 25-35 times in a typical PCR reaction, which generally takes 2-4 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.

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