Gene Disruption in the Yeast Kluyveromyces lactis
Construction of a long flanking mutagenizing cassette based on one-step PCR.
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PROTOCOL
Gene Disruption in theYeast Kluyveromyces lactis ODiLIA QUEIR6 s, MARGARIDA CASAL, PEDRO MORADAS-FERREI RA, and CEciLIA LEAO
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Aim
Construct ion of a long flank ing mutagenizing casse tte based on one-step
rca,
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Introduction
The present method involves th e construction of disrupt ion cassettes and proved to be par ticularly efficient for obtaining null mutants of yeasts (Oueiros et al. 2001). As in the one-step dis ruption method (Rothstein 1983), a first subclo ning of th e genom ic target DNA into a bacterial plasmid is necessary. However, to generate th e deletion and to insert a selecta ble genetic marker, this procedu re does not require restriction sites within th e gene. The protocol involves a pair of primers that are desig ned to am plify th e geno mic DNA regio ns flan king the deletio n, as well as th e bacterial vector (Fig. 1a). Thereinafter, a unique reaction of amplification with two sho rt oligonucleotide pri mers is performed. This step enables one to delimit th e size of th e deletion and of the flanking region s necessary for homo logous recombination. The PCR is carried out with a DNA polym erase,which pr oduces fragments with non-phosphor ylated blunt ends. The product of amplification (Fig. l b) is ligated to a blunt-ended frag ment containing th e Cecilia Leao (:;;;:]), Esccla de Ciencas da Saude, CPl. Universidade do Minho , 4710-057 Braga, Por tugal. em ail: cleao @ecsaude.uminho.p t Odilia Oue iros , IBMC, Universida6e do Porto, 4150·180 Porto, Portugal, ISCSN. 4580 Paredes, Por tugal. email: oqueiros @ibmc.up.pt Pedro Mcradas-Ferrei ra, IBMC, Univers idade do Porto.4150- I80 Porto, Portugal; ICBAS,Universidade do Porto,4100 Porto, Portugal; em ail: [email protected] Margar ida Casal. Centro de Ciencias do Ambiente, Departamento de Biologia, Univers idade do Minho,4710-057 Braga, Por tugal, em ail: mcasal g'blo.uminho.pt
Springer Lab Manual K. Wolf, K. Breun ig, G. Barth (Ed s.) Non-Co nventional Yeasts in Genetics, Biochemist ry and Biotech nology C Springe r-Verlag Berlin Heidelberg 2003
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ODfLlA QUEIR6s et al.
genetic marker. The construction obtained is linearized by rest riction digestion in order to liberate the mutagenizing cassette (Fig..1 c). The method has been successfully used and revealed to be particularly relevant when working with the yeast Kluyveromyces lactis, a species that exhibits low rates of homo logous recombination (Bundock et al. 1999). Other protocols based on PeR-generated DNA fragments containing the marker flanked by short sequences homologous to th e genomic DNA to be deleted have been widely used (for review, see Wach et al. 1998). revealing Iimit ations at the level of homolo gous recombination. Long flanking homologous genera ted protocols increase the transformation efficiency. However, th e prot ocols described so far involve several peR and/or subdoning steps (Wach 1996; Nikawa and Kawabata 1998). The pro cedure described here ha s several advantages: (1) no restriction sites are needed to delimit th e size of the deletion an
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