USE OF VECTORETTE AND SUBVECTORETTE PCR FOR THE ISOLATION OF TERMINAL SEQUENCES FROM Y EAST ARTIFICIAL CHROMOSOME (YAC) CLONES

Development of yeast artificial chromosome (Y AC) vectors, molecular cloning of large segments of chromosomal DNA, and their propagation in yeast cells has become feasible. Overlapping Y AC provides a route to the development of physical maps of entire mammalian chromosomes. A rapid method was developed to isolate and sequence termini of Y AC inserts quickly. The Y AC clone is digested with a range of restriction enzymes, and ligated with a linker at its ends. The digested fragments were amplified using modified vector specific primers and a universal linker primer. PCR products were sequenced and the information used to drive new sets of primers for screening of Y AC libraries to obtain overlapping clones and construct existing Y AC contig.