A strategy for the selection of transcribed sequences in the Xq28 region

Abstract

As an essential step towards an exhaustive analysis of the coding potential of large regions of the genome, we have developed a protocol allowing the rapid isolation of transcripts defined by overlapping clone libraries. The method is based on the hybridisation of cDNA inserts, which had been amplified by PCR from cDNA libraries, to biotinylated DNA from cosmids or cosmid pools. Nonspecific hybrids are then removed, the selected cDNAs are eluted and reamplified by PCR. Using a cosmid containing part of the FMR-1 gene as test, we were able to demonstrate an eighty thousand fold enrichment of cDNAs for this gene after two rounds of selection-amplification. The technique was applied to the analysis of transcripts from two cosmid contigs, together encompassing a region of 900 kb in Xq28. These experiments have thus far resulted in the identification of 81 cDNA clones, of which 54 clones were mapped back to the cosmid contigs. Of the 54 clones placed on the contig maps, 12 cDNA clones can be shown to belong to two genes which have been previously reported (L1CAM and QM).