Rapid and reliable site directed mutagenesis using Kunkel's approach.

Simple item page
dc.contributor.authorHanda, Pen_US
dc.contributor.authorVarshney, Uen_US
dc.date.accessioned1998-04-01en_US
dc.date.accessioned2009-05-27T09:28:07Z
dc.date.available1998-04-01en_US
dc.date.available2009-05-27T09:28:07Z
dc.date.issued1998-04-01en_US
dc.description.abstractOligonucleotide based site directed mutagenesis (SDM) is an invaluable technique in molecular biology. Among the various methods developed for SDM, the PCR-based approach, Kunkel's and the Eckstein's procedures are widely used. The Kunkel's method, on account of its cost effectiveness and simplicity, is preferred by many a scientist. However, a general drawback of this method is the high background due to persistence of the parent template resulting in low efficiency of mutagenesis. In this report, we describe a modification of the Kunkel's method to increase the efficiency of selecting against the wild type strand. We have used Sequenase for the extension reaction, and introduced an in vitro UDG step to enhance the biological selection against the parent strand. Consequently, the efficiency of the modified method is enhanced to allow screening of the mutants directly by DNA sequencing. A step by step single tube protocol which is over in less than three hours makes it a method of choice for efficient and cost-effective site directed mutagenesis.en_US
dc.description.affiliationDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.en_US
dc.identifier.citationHanda P, Varshney U. Rapid and reliable site directed mutagenesis using Kunkel's approach. Indian Journal of Biochemistry & Biophysics. 1998 Apr; 35(2): 63-6en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/27597
dc.language.isoengen_US
dc.source.urihttps://https://www.niscair.res.in/ScienceCommunication/ResearchJournals/rejour/ijbb/ijbb0.aspen_US
dc.subject.meshDNA-Directed DNA Polymeraseen_US
dc.subject.meshEscherichia colien_US
dc.subject.meshGenetic Techniquesen_US
dc.subject.meshIndicators and Reagentsen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshPolymerase Chain Reaction --methodsen_US
dc.subject.meshReproducibility of Resultsen_US
dc.subject.meshTemplates, Geneticen_US
dc.titleRapid and reliable site directed mutagenesis using Kunkel's approach.en_US
dc.typeJournal Articleen_US
dc.typeResearch Support, Non-U.S. Gov'ten_US
Files
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.79 KB
Format:
Plain Text
Description:
Download
Collections
Indian Journal of Biochemistry & Biophysics