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Polymerase chain reaction. Who would have thought a bacterium hanging out in a hot spring in Yellowstone National Park would spark a revolutionary new laboratory technique? The polymerase chain reaction, now widely used in research laboratories and doctor's offices, relies on the

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Polymerase chain reaction
Polymerase chain reaction

Who would have thought a bacterium hanging out in a hot spring

in Yellowstone National Park would spark a revolutionary new

laboratory technique? The polymerase chain reaction, now widely

used in research laboratories and doctor's offices, relies on the

ability of DNA-copying enzymes to remain stable at high

temperatures. The polymerase from Thermus aquaticus (Taq),

a bacterium from Yellowstone can produce millions of copies of

a single DNA segment in a matter of hours. In nature, most organisms

copy their DNA in the same way. PCR mimics the natural process,

only it does it in a test tube. When any cell divides, enzymes called

polymerases make a copy of all the DNA in each chromosome.

The first step in this process is to "unzip" the two DNA chains of

the double helix. As the two strands separate, DNA polymerase

makes a copy using each strand as a template.


The role of the primer
The role of the primer

To copy DNA, polymerase requires two other components:

1. a supply of the four nucleotide bases

2. a primer.

DNA polymerases, whether from humans, bacteria, or

viruses, cannot copy a chain of DNA without a short

sequence of nucleotides to "prime" the process, or get it

started. So the cell has another enzyme called a primase

that actually makes the first few nucleotides of the copy.

This stretch of DNA is called a primer. Once the primer

is made, the polymerase can take over making the rest of

the new chain.


Step i dna melting
Step I: DNA melting

The three parts of the polymerase chain reaction are

carried out in the same vial, but at different temperatures.

The first part of the process separates the two DNA

chains in the double helix. This is done simply by

heating the vial to 95 oC for 30 seconds.

double-stranded DNA single-stranded DNA


Step ii primer annealing
Step II: Primer annealing

The primers cannot bind to the DNA strands at such a

high temperature, so the vial is cooled to 55 oC. At

this temperature, the primers bind or "anneal" to the

appropriate location in the DNA strands.

This takes about 20 seconds.

single-stranded DNA + primer annealed DNA

The final step of the reaction is to make a complete

copy of the templates. Since the Taq polymerase works

best at ca. 75 oC, the temperature of the vial is raised.


Step iii primer extension
Step III: Primer extension

The Taq polymerase begins adding nucleotides to the

primer and eventually makes a complementary copy

of the section of the template that lies between the

primers. This completes one PCR cycle.

primer-annealed DNA primer-extended DNA

with Taq polymerase and

dATP, dTTP, dGTP, dCTP


Thermal cycling
Thermal cycling

At the end of a cycle, each piece of DNA in the vial

has been duplicated. Since the cycle can be repeated

30 or more times and each newly synthesized DNA

piece can act as a new template, one can obtain 230 or

ca. 1 million copies of a single piece of DNA.

amplified region

Note that the region of the DNA between the two

primers will be amplified. The flanking sequences

not. The entire process takes about three hours.


Pcr amplification
PCR amplification

The figure on the left shows the series of steps

in a single cycle. The exponential growth of

the double helical segment between the two

primers is illustrated above.


Taq polymerase
Taq polymerase

Taq Polymerase In Complex With Tp7, An Inhibitory Fab


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