EFFICIENCY AND SPECIFICITY OF GENE ISOLATION BY EXON AMPLIFICATION

Exon amplification is an increasingly popular approach to the identifi cation of transcribed sequences and will complement other strategies t o isolate genes. We have used this system to amplify candidate exons f rom 32 cosmids, including 8 cosmids which span a well characterized 18 5-kb region of the human major histocompatibility class II region on C hromosome (Chr) 6. We have examined the efficiency, specificity, and r eproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and freq uency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes test ed (including exons which are differentially spliced) and obtained art efacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of non-coding sequences flanked by regio ns with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.