PCR with Random Primers to Obtain Sequence from Yeast Artificial Chromosome Insert Ends or Plasmids

Abstract

A technique that can be used to isolate vector/insert junctions from clones in vectors, such as yeast artificial chromosomes and PIs, and to sequence plasmid inserts more rapidly has been developed. A vector primer is combined with single, randomly chosen oligonucleotides in PCRs, to create pools of products. With 12–24 random primers used in separate reactions, a given insert junction can frequently be isolated. For plasmid inserts, multiple products are created that can be sequenced from their random-primed ends to provide internal coverage for a clone. It is often possible to sequence a significant portion of an insert with one set of reactions. The speed and simplicity of the method in each case and its use of existing techniques and reagents make it appealing.