Tuesday, March 24, 2009

PCR Components

The Polymerase Chain Reaction (PCR) created a revolution in molecular biology research and its applications. PCR is an in vitro method that enzymatically amplifies specific DNA sequences using oligonucleotide primers that flank the region of interest in the target DNA. The principle involves a repetitive series of cycles each of which consist of template denaturation, primer annealing, and extension of the annealed primers by a DNA polymerase to create the exponential accumulation of a specific fragment whose ends are determined by the 5’ ends of the primers. The PCR is so named because it involves a polymerase and the products synthesized in each cycle can serve as templates in the next so the number of DNA copies approximately doubles at every cycle to create a chain reaction similar to the principles in a nuclear reactor.

Polymerase enzyme used in the PCR reaction is isolated from Thermus aquaticus, therefore it is named Taq polymerase. Taq polymerase is a thermostable enzyme that enabled the amplification reaction to be carried out by cycling the temperature within the reaction tube after mixing all the reaction components.

The taq polymerase enzyme works with high efficiency in relatively simple buffer systems. Thus the essential components of the polymerase chain reaction include: the DNA template, two defined oligos to act as primers, the four deoxynucleotide triphosphates (dNTPs) for incorporation into the new DNA strands, Taq polymerase and magnesium, which is the cationic cofactor of the enzyme, in a Tris buffer of appropriate pH and salt concentration. The salt most frequently used is potassium chloride. A layer of light mineral oil is always placed on top of each reaction to prevent evaporation which would otherwise stop the reaction within a few cycles.

Those basic components remain largely unchanged for most applications of PCR, but their concentrations and the buffering pH of the reaction vary according to the type of experiment, the nature of the primers and/or template, and the experience of the experimenter. The reaction mix of the PCR (the concentrationand pH ranges) can see in the following table.

PCR components

You can also use the standard PCR component, here is the list:

10 mM Tris-HCl pH 8.4

50 mM KCl

1 mM Magnesium chloride

0.05% (v/v) Tween 20

0.2 mM dNTPs

100 pmol each primer

1 U Taq polymerase


Reference: Number 4 on References