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Yeast. 2011 Mar;28(3):205-12. doi: 10.1002/yea.1831. Epub 2010 Nov 22.

Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach.

Author information

1
Temasek Life Sciences Laboratory, National University of Singapore, Republic of Singapore. tangxie@tll.org.sg

Abstract

A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination.

PMID:
21360732
DOI:
10.1002/yea.1831
[Indexed for MEDLINE]
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