title: Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach.
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.
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Republic of Singapore. tangxie@tll.org.sg
authors.forename: Xie
authors.initials: X
authors.lastname: Tang
authors.forename: Junqi
authors.initials: J
authors.lastname: Huang
authors.forename: Anup
authors.initials: A
authors.lastname: Padmanabhan
authors.forename: Kavya
authors.initials: K
authors.lastname: Bakka
authors.forename: Yun
authors.initials: Y
authors.lastname: Bao
authors.forename: Brenda Yuelin
authors.initials: BY
authors.lastname: Tan
authors.forename: W Zacheus
authors.initials: WZ
authors.lastname: Cande
authors.forename: Mohan K
authors.initials: MK
authors.lastname: Balasubramanian
mesh_terms: D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D005827:Genetics, Microbial / Q000379:methods*; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000502:physiology
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins