Polymerase Chain Reaction (PCR)

Definition:
Amplification means making multiple identical copies (replicates) of a DNA sequence. This can be carried out by various methods, that include in vivo amplification including cell cloning where host cells (manipulated using a vector to contain a DNA insert of interest) are allowed to divide and, as they do so, the insert is replicated.


Historical background…

• Method first proposed by H. G. Khorana & colleagues in 1970’s.
• 15 years later the idea was independently conceived by Karry Mullis in 1983.
• Used the Klenow fragment of E. coli DNA polymerase to describe the in-vitro amplification of genes.
• Saiki et al in 1988 used the thermostable DNA polymerase from Thermus aquaticus and greatly increased the efficiency of PCR.
• In 1989, Science magazine selected PCR as the major scientific development and Taq DNA polymerase as the molecule of the year.
• Karry Mullis was awarded the Noble price for chemistry in 1993.

Seven essential components required

1. Template DNA
2. A thermostable DNA polymerase
3. A pair of synthetic oligonucleotide primers.
4. Divalent cations (Mg 2+ )
5. dNTPs
6. Buffer to maintain pH ( Tris-Cl pH 8.3 – 8.8)
7. Monovalent cations
   
   The PCR usually consists of a series of 30 to 35 cycles. Most commonly, PCR is carried out in three steps, often preceded by one temperature hold at the start and followed by one hold at the end. A typical PCR cycle has following steps

ØDenaturation (94-95°C, for ~ 30 s)

The template is denatured by heat

ØAnnealing (55-60°C, for ~ 30 s)

Annealing of oligonucleotide primers to single stranded       target sequences

ØElongation (72°C)

Extension of annealed primers by a thermostable  polymerase