Introduction of Polymerase Chain Reaction

Polymerase is a method deleveoped by Kary Mullis in the 1980s. The PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. The reason being is that DNA polymerase can add a nucleotide only onto a pre-existing 3'-OH group, it needs a primer- "copier" to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the research wants to amplify. At the end of the PCR reaction, the specific sequence will be accumlated in the bigger copies.

How it works?



Components needed for PCR reaction to takes place:

1) DNA template: the sample DNA that contains the target sequence. At the beginning of the reaction, the high temperature is applied to the double stranded DNA molecule in order to seperate the strands from each other.

2) DNA polymerase: An enzyme that synthesize new strands of DNA complementary to the target sequence. The first and commonly used of these enzymes is Taq DNA polymerase (from Thermis aquaticus). Pfu DNA polymerase is widely used due to its high feidelity in copying the DNA. Although these enzymes are subtly different, both have the capabilities which make them suitable for PCR. First, they can generate new strands of DNA using a DNA template and prmers. Secondly, they are heat resistants.

3) Primers- a short pieces of single stranded DNA that are compementart to the target sequence. The polymerase starts synthesizing new DNA from the end of the thyme.

4) Nucleotides- single units of the baseses A, T, G and C which are known as the "building blocks" for new DNA strands.



Limitation:

The PCR reaction starts to generate copies of the target sequence exponentially. During the exponential phase of the reaction, it is impossible to extrapolate back to determine the starting quantity of the target sequence which contain in the sample. Due to the presence of inhibitors found in the samples, reagent limitation, accumaltion of the pyrophospahe molecules, and self-annealing of the accumlatin product, the PCR reaction simply ceases to amplify targate sequence at an exponential rate and a "plateau effect" occurs and thus making the end point quantification of PCR product unreliable.

Videos on Polymerase Chain Reaction




Reference:

NCBI(n.d.). Polymerase Chain Reaction. http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml. Retrieved date: 10 June 2010.

Grohmann, L., & Mäde, D. (2009). Detection of genetically modified rice: collaborative validation study of a construct-specific real-time PCR method for detection of transgenic Bt rice. European Food Research & Technology, 228(3), 497-500. doi:10.1007/s00217-008-0964-1.

Youtube(2010). PCR reaction. Website: http://www.youtube.com/watch?v=_YgXcJ4n-kQ. Retrieved date: 11 July 2010

Posted By: Nicholas Tan