POLYMERASE CHAIN REACTION; AN EFFECTIVE DIAGNOSTIC AND RESEARCH TOOL.

Brief History

PCR technique was invented by Kary Mullis in 1985 while working?as a Chemist at Cetus Cooperation, a biotechnology firm in California.

Before PCR?

Cloning; a technique used to create exact genetic replica of another cell, tissue or organism. Cloning was too laborious which brought about the invention of PCR.

What is PCR?

It is a technique used to amplify or make many copies of small segments of DNA in vitro. It is also called a Molecular Photocopy Machine.

Why is PCR necessary?

For downstream reactions when more DNA is needed. It’s able to amplify a specific or targeted segments of DNA mixture.

What Purpose?

To amplify millions of double stranded DNA molecules of same size and sequence by an enzymatic method and cycling condition.

Key requirements for PCR

• Primers: These are short oligonucleotides usually 18-22bp in length, serves as the starting material for amplification, and binds to complementary sequence on the DNA template.
• Thermo-stable DNA polymerase enzyme: e.g Taq polymerase which can’t be inactivated by heating up to 95C-DNA.
• Thermal Cycler: This is the photocopying machine, already programmed to carry out heating and cooling of samples over a number of cycles.
• Template DNA: It is the DNA?that the DNA polymerase reads and copies. Quality is very important, must remain pure and intact.
• Nucleotides: These are monomers of DNA acting as building blocks for making DNA copies. Other components include buffer, water and MgCl2.

Steps in PCR

• Denaturation: This is the unwinding of the double stranded DNA into single strands. (@90-95?°C).
• Annealing of Primers: Primers binds to the complementary sequence on the targeted DNA. (@ 40-60°C).
• Elongation / Extension: Primers extend by the DNA polymerase and synthesize a complementary strand. (@70-74°C). And this process continues until millions of DNA is being amplified.

NOTE: At each PCR step, there is either a rise or fall in temperature.

Types of PCR?

1. Nested PCR

• Uses two set of primers in two successive PCR steps.
• Useful in amplifying DNA at very low concentrations.
• Requires more detailed knowledge of the targeted sequence.

2. Multiplex PCR

• Uses multiple set of unique primers.
• Produces amplicon of varying sizes specific to different DNA sequences.
• Targets multiple genes, pathogens at once.

3. Real Time PCR

• Highly quantitative.
• Requires the use of DNA binding dyes.
• Determines level of gene expression. RT-PCR Identifies known sequence from a cellular or tissue RNA.
• Uses reverse transcriptase enzyme to synthesize cDNA from RNA.

4. Mutation PCR

• Detects specific mutation (SNPs) in the region of interest.
• Requires reactions to be carried out in duplicates.

Optimization tips in PCR?

• Ensure template purity.
• Accurate and specific primer selection.
• Appropriate annealing temperature.

Applications of PCR

• Molecular and epidemiological studies.
• Detection and diagnosis of infectious diseases (Lassa virus, HIV, TB, Ebola).
• Sequencing; bioinformatics, genomic cloning.
• Genetic engineering; gene expression studies.