title: Nucleotide sequencing double-stranded plasmids with primers selected from a nonamer library.
abstract: 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.
authors: Molecular Biology Unit 7242-267-510, Upjohn Company, Kalamazoo, MI 49007.
authors: J L
authors: JL
authors: Slightom
authors: J H
authors: JH
authors: Bock
authors: D R
authors: DR
authors: Siemieniak
authors: G D
authors: GD
authors: Hurst
authors: K L
authors: KL
authors: Beattie
mesh_terms: D017931:DNA Primers; D015723:Gene Library; D005914:Globins / Q000235:genetics; D006801:Humans; D010957:Plasmids; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D005914:Globins