Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

✍ Scribed by Thomas W. Okita

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 886 KB
• Volume: 144
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90107-1

✦ Synopsis

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions SO-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Ba131 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M 13 mpl0 or mpl I. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.