Polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or a few copies of a piece of DNA to produce thousands to millions of copies of a particular DNA sequence within short time.

The technique was developed in 1983 by Kary Mullis.

A typical PCR involves 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps.

Initialization step

This step consists of heating the reaction to a temperature of 94–96 °C . It is required for activation of DNA polymerase.

Denaturation Steps

This step consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template .

Annealing Step

The temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template.

Extension/Elongation step

At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand polymerizing at a rate of e a thousand bases per minute.At each extension step, the amount of DNA target is doubled, leading to exponential  amplification.

The temperature of the step varies according to type of enzyme used.

Sometimes, another step called final elongation is performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure full extension of any remaining single-stranded DNA

Final hold

At 4–15 °C . For short-term storage of the reaction.

Applications and Uses Of PCR

  DNA isolation

Becasue the test allows selective amplification of a specific region of DNA,  PCR itself is used to augment many other tests where supply of the DNA is required . These include  hybridization and DNA cloning, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism.

Bacterial colonies (E. coli) can be rapidly screened by PCR for correct DNA vector constructs.

Amplification and quantification of DNA is  very important in Forensic analysis where trace amount of DNA may be available for study.

Disease Diagnosis

• Early diagnosis of malignant diseases such as leukemia and lymphoma
• Identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses

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