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Wednesday, May 1, 2013

PCR & Site Directed Mutagenesis


PCR – Polymerase chain Reaction

Polymerase chain reaction or PCR is a powerful tool for amplifying a stretch of DNA.From a single piece of target DNA one can make enough copies for sequencing, cloning or for gel electrophoresis.
PCR is performed generally in 0.2 or 0.1ml tube in a thermo cycler, a machine which maintains temperature as per the users input. The PCR reaction mixture or the master mix will have the following components:
  • Target DNA; Isolated from either of these samples :infected Blood or sputum, Tissues, Hair, etc.
  • Primers – Specific to the target DNA to be amplified.
  • dNTPs
  • Taq DNA polymerase
  • Some times PCR enhancers like DMSO, BSA, etc.
Polymerase chain reaction has 3 steps:
  1. Initial Denaturation: Temperature is raised to above 90oC, which breaks Hydrogen bonding between the two strands resulting in strand separation
  2. Annealing:In this step temperature is lowered resulting in the binding of primers to its complementary sequence in the target DNA.
  3. Extension: Here in this step temperature is raised to 72oC there by Taq polymerase adds up the nucleotides to the primers in 5’ to 3’ direction.
DNA duplex is produced after this process, this continues for 40 cycles. One copy becomes two, two becomes 4, and so on.
The no. of copies generated after n cycles are given by the general formula 2n.
if there are X copies in the beginning and after n cycles it will be X*2n copies.

Site Directed Mutagenesis

The primer used in the reaction mixture do not have to match exactly they can still anneal with few number of point mutations which the base pairs are not complementary to the target DNA.
Thus the amplified products will have the mismatched sequence, over the no of cycles proceeds there will be more copied of DNA which has the new sequence in it than from the original template DNA.
This is a convenient way of making changes to the DNA. If the new sequence or the altered sequence is coding for protein then the same amplified product can be cloned and expressed.

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