Polymerase Chain Reactio for Site Directed Mutagenesis, DNA Sequence, Mutations, Gene Sequence, Taq Polymerase

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Home >> Biotechnology and Genomics >> Polymerase Chain Reaction-PCR and Gene Amplification >> Polymerase Chain Reaction of Site Directed Mutagenesis

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PCR for site directed mutagenesis

This technique is used for introducing mutations at the desired place in a DNA sequence by altering the sequences of primers. Since mutations are introduced only through primers, mutations are limited to the ends of the gene sequence. A variation of this technique allows mutations to be introduced at any place of interest in the gene. The method is described as overlap extension, which works as follows:

Site Directed Mutagenesis using PCR

Site Directed Mutagenesis using PCR (minor changes are introduced in primers; these minor changes do not interfere with PCR

In two separate PCR reactions, a particular gene is amplified into two separate segments. In each of the two reactions, there is one primer at the end of the gene and the other (with desired alteration for mutation) internal to the sequence. The internal modified primers in two reactions are complementary to one another, so that the amplified products will have their ends internal to the original sequence. These internal ends of products in two reactions will overlap, so that if the two PCR products are mixed and used again for PCR, their internal ends function as primers. Extension of these primers by Taq polymerase results in the formation of a complete gene, with mutations incorporated at internal sites

Introduction of a Mutation at an Internal Site using Overlap Extension

Introduction of a Mutation at an internal site using overlap extension(two pairs of primers A & B and C & D are used. B and C represent internal sites and overlap each other

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