Biology Forums - Study Force

1. Describes the steps of how a PCR generates many copies of a particular sequence.

2. How and why are positive and negative controls used for the PCR?

3.What temperatures are involved in the PCR cycle?

4. What processes take place at each temperature?

5.How does gel electrophoresis sort DNA fragments?

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.

Explanation:

The basic steps of PCR are:

Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
Annealing (55-65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

Requirements of PCR:

Thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.

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).

PCR primer

PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides long. 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.

A negative control is used to ensure that no such contamination occurred. If there is no contamination, the negative control should not produce DNA.The positive control uses another DNA template that has been successfully amplified by the given primers in the past.

If the PCR does not amplify the sample DNA but does amplify the positive control, there is a problem with the sample DNA. If the PCR does not amplify the sample DNA or the positive control, there is a problem with the PCR process.