Biotechnology and Bioinformatics (1+1)

4.1.1. Introduction

Polymerase chain reaction (PCR) helps in gene rating numerous copies of DNA from a small initial sample.

The polymerase chain reaction (PCR) process was discovered in 1983 by Kary Mullis who was awarded the Nobel Prize for chemistry in 1993.

• Polymerase chain reaction is basically a technique that allows the selective amplification of any fragment of DNA, provided the DNA sequence s flanking the fragment are known.
• The system works so well because amplification of a target DNA sequence is exponential.
• Each heating and cooling cycle results in the doubling of the amount of template, hence after 20 cycles the yield of PCR product is approximately one million copies of the single target DNA molecule.
• The original procedure used the DNA polymerase I Klenow fragment from E. coli. This had the drawback that between each cycle the DNA had to be denatured (94°C) and new enzyme added.
• To circumvent this problem thermostable DNA polymerase isolated from the bacteria Thermus aquaticus YT1, which grows in the hot springs of Yellowstone National park is used.
• The enzyme works optimally at 72°C and can also withstand heating to 94°C for short periods of time.
• This means that during 20 cycles of PCR the enzyme does not have to be replenished.

PCR amplification requires

• two oligonucleotide primer s, selective primer and reverse primer,
• four dNTPs (deoxy nucleotide triphosphates),
• magnesium ions in molar excesses of the dNTPs and
• a thermostable DNA polymerase to perform DNA synthesis. The quantities of oligonucleotide primers, dNTPs, and Mg++ may vary for each specific application. The conditions need to be optimized for different DNA fragments and oligonucleotide primers.