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Title: Modification of overlap extension PCR: A mutagenic approach
Authors: Patel, Darshan H
Wi, Seung Gon
Bae, Hyeun Jong
Keywords: DNA polymerase;ex taq;overlap extension PCR;site directed mutagenesis;one step overlap extension-PCR
Issue Date: Apr-2009
Publisher: CSIR
Abstract: In vitro site-directed repair or creation of a mutation is an invaluable technique in genetic and protein engineering. Several methods have appeared in literature but still require many modifications. We describe a rapid and efficient modified overlap extension PCR method for multiple uses in mutagenesis studies. The protocol is based on two rounds of PCR with the help of two sets of primers, two flanking and two internal mutagenic primers. Two fragments of DNA prepared in first round of PCR are then allowed themselves to anneal in the second stage of PCR using gradient annealing temperature without using flanking primers. This protocol has been used for correcting a mutation caused in exoglucanase (CBHII) gene of Trichoderma spp. We successfully synthesized the full length of gene from two fragments in the second round of PCR in lesser time.
Page(s): 183-186
ISSN: 0972-5849
Appears in Collections:IJBT Vol.08(2) [April 2009]

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