Nucleotide sequencing double-stranded plasmids with primers selected from a nonamer library.

Nonamer primers, selected from a nonamer library, were tested by sequencing two plasmid subclones containing known insert sequences. These sequences were scanned (nonamer-mapped) against the 2391-member nonamer library to identify all members that share a 100% match at only one site. A total of 59 nonamers were tested using a slightly modified T7 polymerase sequencing procedure for double-stranded DNA. The success rate for nonamer primed reactions was about 60%, and single-stranded coverage was obtained for approximately 90% of each plasmid insert. The results presented demonstrate that a nonamer library, with as few as 2391 members, can greatly aid the completion of many sequencing projects by reducing the number of required custom primers. With the development of a technique for the rapid identification of all useful library primers for a particular sequencing project, one could envision a high-throughput shotgun-type sequencing procedure that would not require large numbers of subclones.