Isolation of specific clones from nonarrayed BAC libraries through homologous recombination.

ABSTRACT: We have developed a new approach to screen bacterial artificial chromosome (BAC) libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380) with temperature inducible homologous recombination (HR) capability. We amplified one library segment, induced HR at 42°C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The adva

SUBMITTER: Nefedov M 

PROVIDER: S-EPMC2957146 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature