Polymerase Chain Reaction (PCR) - Amplification of DNA Samples
What is PCR?
Polymerase chain reaction
, or PCR, is a process by which small samples of DNA are made larger, or amplified. By using PCR, labs are able to
create larger batches of DNA from small samples for easier analysis.
How does it work?
A sample of DNA is collected and exposed to the "PCR mix," a solution of primers, free nucleotide bases, and DNA polymerase in a buffer.
Each of these components plays an important part in ensuring that the DNA is amplified.
There are three basic steps in PCR:
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Denaturing: Separating the DNA Strands
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Annealing: Attaching the PCR Primers
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Elongation: Rebuilding the DNA Strands
1. Denaturing: Separating the DNA Strands
The first step of PCR is denaturing the DNA sample. The DNA is heated to approximately 95 degrees Celsius,
causing the DNA to unwind from its normal shape and the two backbone strands to separate.
At the end of this step, the lab has two single strands of the DNA backbone for each molecule of the original DNA.
2. Annealing: Attaching the PCR Primers
After the DNA is denatured, the sample is cooled to approximately 60 degrees Celsius and molecules called primers bind to the two single DNA strands.
The primers prepare the strands to reform into complete DNA molecules.
3. Elongation: Rebuilding the DNA Strands
Once the primers have attached to the single DNA strands, the sample is heated to approximately 72 degrees Celsius and the DNA polymerase
is activated. The polymerase recruits free DNA bases in the sample solution, matches them to the single DNA strand, and extends the primer.
This process, called elongation, results in a double-stranded copy of the original DNA sample.
At the end of these three steps, the lab has two copies of the original DNA sample.
This three-step process is repeated at least 30 more times, ultimately resulting in millions of copies of the original DNA
sample.
Why use PCR?
Most DNA testing laboratories now use PCR, because it allows them to make many copies of the samples, reducing the amount of time necessary for
analysis. With other methods, such as RFLP, larger samples are needed. With PCR, labs can copy a small sample many times to create a larger batch of
sample copies for easier analysis. Essentially, the more copies of the sample they have, the faster they can complete the DNA test.
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