SCANNING FOR GENES IN LARGE GENOMIC REGIONS - COSMID-BASED EXON TRAPPING OF MULTIPLE EXONS IN A SINGLE-PRODUCT

To facilitate the scanning of large genomic regions for the presence o f exonic gene segments we have constructed a cosmid-based exon trap ve ctor. The vector serves a dual purpose since it is also suitable for c ontig construction and physical mapping. The exon trap cassette of vec tor sCOGH1 consists of the human growth hormone gene driven by the mou se metallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenn e Muscular Dystrophy gene. All exons present in the inserts were succe ssfully retrieved and no cryptic products were detected. Up to seven e xons were isolated simultaneously in a single spliced product. The sys tem has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped pro ducts, using a combination of tailed PCR primers directing protein syn thesis in three different reading frames, followed by in vitro transcr iption-translation. Having larger stretches of coding sequence in a si ngle exon trap product rather than small single exons greatly facilita tes further analysis of potential genes and offers new possibilities f or direct mutation analysis of exon trap material.