Vector–Hexamer PCR Isolation of All Insert Ends from a YAC Contig of the MouseIgh Locus

Abstract

We have developed a simple PCR strategy, termed vector-hexamer PCR, that is unique in its ability to easily recover every insert end from large insert clones in YAC and BAC vectors. We used this method to amplify and isolate all insert ends from a YAC contig covering the mouse Igh locus. Seventy-seven ends were amplified and sequenced from 36 YAC clones from four libraries in the pYAC4 vector. Unexpectedly, 40% of the insert ends of these YACs were LINE1 repeats. Nonrepetitive ends were suitable for use as probes on Southern blots of digested YACs to identify overlaps and construct a contig. The same strategy was used successfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBAC11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investigation.[The nucleotide sequence data reported in this paper are accessible in GenBank under accession nos. B07512-B07598.]

A Three-Megabase Yeast Artificial Chromosome Contig Spanning the C57BL Mouse Igh Locus

“…In the V H gene array, we found another potential impediment to YAC stability, an extraordinary frequency of LINE1 elements. In our sequencing of YAC insert ends, we found that 40% belonged to the L1 family of high copy number repetitive sequences (16); this frequency is surprisingly higher than the 5% incidence observed in another YAC contig on chromosome 11 (40), but more similar to the 25% incidence in the Igk locus (41) and a probably higher frequency in the MHC (42). Interestingly, the human IGH locus also has a 40% content of L1 sequence (1).…”

“…For this study, we developed a new generalized strategy, called vector-hexamer PCR, for the isolation of insert ends from YACs (16). YAC insert ends were isolated from all 38 clones to characterize YAC overlap and to detect chimeric clones.…”

“…YAC insert ends were prepared for hybridization probes by reamplifying third-round gel purified ends with the innermost nested primer to reduce the content of the vector arm in the PCR product. Amplification products were electrophoresed on acrylamide gels and the correct size bands were isolated (16). Depending upon the brightness of the band, 1-10 microliters of gel eluate were labeled for use as probes.…”

“…Both ends of each YAC were isolated by the vector-hexamer technique (16). This method consists of the PCR amplification of YAC insert ends using a nested series of vector primers in conjunction with a hexamer primer that anneals to a short arbitrary sequence randomly located in the mouse insert.…”

“…Several additional YACs were obtained from a fifth library through reference to Internet-posted data (15). YAC insert ends were isolated from all clones by vector-hexamer PCR (16) to characterize YAC overlap and assemble the contig, and to detect chimeric clones. This physical YAC map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.…”