Wednesday, August 17, 2011

Polymerase Chain Reaction (PCR)

PCR is a technique widely used in molecular biology. It derives its name from one of its key components a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses the DNA generated is used as a template for replication.With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude generating millions or more copies of the DNA piece. PCR can be extensively modified to perform

a wide array of genetic manipulations ( Bartlett & Stirling , 2003) .

Almost all PCR applications employ a heat-stable DNA polymerase such as Taq polymerase which is isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks.The nucleotides by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers) are required for initiation of DNA synthesis. The vast
majority of PCR methods use thermal cycling by alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as the template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions ( Pavlov et al ., 2004).

The basic PCR set up requires several components and reagents . These components include:

*DNA template that contains the DNA region (target) to be amplified.
*Two primers, which are complementary to the DNA regions at the 5' or 3' ends of the DNA region.
*Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C. *Deoxynucleoside triphosphates (dNTPs; or deoxynucleotide triphosphates) are the building blocks from which the DNA polymerases synthesizes a new DNA strand.
*Buffer solution is providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.

*Divalent cations, magnesium or manganese ions.Generally Mg2+ is used but Mn2+ can be utilized for PCR-mediated DNA mutagenesis as higher Mn2+ concentration increases the error rate during DNA synthesis (Pavlov et al ., 2004 ).

According to Rychlik et al .,( 1990) each PCR cycle which results in exponential amplification of the sequences between the two primer binding sites consists of several steps :

**Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used) which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR ( Sharkey et al ., 1994).

**Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands yielding single strands of DNA ( Sharkey et al ., 1994).

**Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template.Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis (Chien et al ., 1976).

**Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand

complementary to the DNA template strand by adding dNTPs in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. At optimum temperature , the DNA polymerase will polymrize a thousand bases per minute. Under
optimum conditions if there are enough substrates and reagents, at each extension step the amount of DNA targets are doubled leading to exponential (geometric) amplifications of the specific DNA fragment (Pavlov et al ., 2006).

**Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended (Lawyer et al ., 1993).
**Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction (Lawyer et al ., 1993).
b)- PCR stages :)
According to Cheng et al., (1994), the PCR process can be divided into three stages:

**Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.

**Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

**Plateau: No more product accumulates due to consumption of reagents and enzyme.

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