Isolation of Coding Sequence from Cosmids and YACs by Exon Amplification

Exon amplification is an increasingly popular approach to the identification of transcribed sequences and complements other strategies to rapidly isolate coding sequence. The following chapter describes application of this system to amplify candidate exons from cosmids and YACs, including 8 cosmids which span a well characterised 185 kb region of the human major histocompatibility class II region on chromosome 6. We have examined the efficiency, specificity and reproducibility of the system in isolating exons from genes on cosmids and YACs and describe the nature and frequency of artefact amplifications in routine screening. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids and YACs which have different numbers of genes and gene structures and describe an efficient approach to the isolation of full length cDNA sequences by the integration of exon amplification and cDNA enrichment technology.