VECTOR-HEXAMER PCR ISOLATION OF AII INSERT ENDS FROM A YAC CONTIG OF THE MOUSE IGH LOCUS

We have developed a simple PCR strategy, termed vector-hexamer PCR, th at is unique in its ability to easily recover every insert end from la rge insert clones in YAC and BAC vectors. We used this method to ampli fy and isolate all insert ends from a YAC contig covering the mouse Ig h locus. Seventy-seven ends were amplified and sequenced from 36 YAC c lones From four libraries in the pYAC4 vector. Unexpectedly, 40% of th e insert ends of these YACs were LINE1 repeats. Nonrepetitive ends wer e suitable for use as probes on Southern blots of digested YACs to ide ntify overlaps and construct a contig. The same strategy was used succ essfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBA C11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investig ation.