NUCLEOTIDE SEQUENCING DOUBLE-STRANDED PLASMIDS WITH PRIMERS SELECTED FROM A NONAMER LIBRARY

Nonamer primers, selected from a nonamer library, were tested by seque ncing two plasmid subclones containing known insert sequences. These s equences were scanned (nonamer-mapped) against the 2391-member nonamer library to identify all members that share a 100% match at only one s ite. A total of 59 nonamers were tested using a slightly modified T7 p olymerase sequencing procedure for double-stranded DNA. The success ra te for nonamer primed reactions was about 60%, and single-stranded cov erage was obtained for approximately 90% of each plasmid insert The re sults presented demonstrate that a nonamer library, with as few as 239 1 members, can greatly aid the completion of many sequencing projects by reducing the number of required custom primers. With the developmen t of a technique for the rapid identification of all useful library pr imers for a particular sequencing project, one could envision a high-t hroughput shotgun-type sequencing procedure that would not require lar ge numbers of subclones.