PCR Mutagenesis: Overlap Extension

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Polymerase Chain-Reaction (PCR) has become the backbone of most Methods in Molecular Biology and site-specific mutagenesis no exception. The key to PCR-based mutation of DNA is careful design of primers. In the simplest case, a point-mutation can be inserted into all PCR products by adding a point mutation to all primers. Unfortunately, for linear DNA, this method only works for mutagenesis at the ends of the template (where the primers bind).

Overlap extension, is a powerful 2-step, multi-PCR technique that can insert mutations at any position and of any size (including whole deletions or insertions). It accomplished this using chimeric primers to (1) cut out pieces of DNA and (2) reassemble them at overlap points:

  1. Primary PCR: Two parallel PCR reactions are run to cut out the pieces of DNA surrounding the insertion or deletion site. Each reaction has two primers: a normal set (A & D) and a chimeric set (C & D) that include an extra ~9 bases that will control reassembly. (chimeric primers = ~18 normal bases + ~9 overlapping bases)

  2. Ligation PCR: The two “overlapping pieces” from the “Primary PCR” are combined with the “normal set” of primers (A & D) for the final PCR step. First, ligation occurs as the “overlapping pieces” template synthesis of the final product; Second, primers A & D amplify the synthesis of the final product.

 

REFERENCES:

  1. Green, M.R.; Sambrook, J. Molecular Cloning: A Laboratory Manual 4th Ed., 2012, Cold Spring Harbor Laboratory Press
  2. Alberts, B. Molecular Biology of the Cell 5th Ed. Garland Science 2008
  3. Murphy, K. Janeway’s Immunobiology 8th Ed. Garland Science 2012

 

Creative Commons License
This work by Eugene Douglass and Chad Miller is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

 

Creative Commons 

License
This work by Eugene Douglass and Chad Miller is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

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