Rapid isolation of genes from an indexed genomic library of C. cinereus in a novel pab1 + cosmid

Abstract

In this study we present an indexed genomic library of homokaryon AmutBmut constructed within a novel cosmid carrying pab1 + as a selectable Coprinus marker. The average insert size per cosmid comprises 41 kb. We screened the library and detected copies of known ( a1–2, β-tub, cgl1, ras, trp1) and of new Coprinus genes ( cac, lac1, lac2, lac3). Screening was performed either by Southern blot hybridisation or more efficiently by non-radioactive PCR amplification. We successfully applied PCR with specific and with degenerate primers, multiplex PCR and colony PCR in library screening. Our results suggest a new, more efficient pooling strategy for future high throughput screenings to be used in PCR with pooled cosmid DNAs, or in a less laborious approach using pooled Escherichia coli colonies for PCR.