Plasmid DNA sequencing using highly degenerate oligonucleotides as primers.

Sequencing double-stranded DNA templates has become an efficient procedure (1) being much less time-consuming than the preparation and subsequent sequencing of inserts in phage singlestranded DNA. The ability to use highly degenerate oligonucleotides instead of sequence-specific primers would make this method even more powerful for the rapid sequencing ofDNA that codes for a protein for which amino acid sequence information is already available. Here I report the applicability -0 of this method, using oligonucleotides of almost 1000-fold degeneracy (i.e. only one out of a mixture of 1000 oligomers has the correct base sequence) at appropriate primer:template ratios, to directly start sequencing within the gene coding for S. cerevisiae guanylate kinase, designated GUKI, whose amino acid sequence has recently been published (2). Two mixed oligonucleotide probes were prepared which corresponded to the amino acid sequence from position 61 to 70 (Oligo I: 5'-CCA (T, C) TC (A, G, T) AT (A, G) AA (T, C) TC (A, G) TT (A, G) TT (T, C) TT (A, G, T) AT CAT-3'), and from 179 to 186 (Oligo II: 5'-(T, C) TT (T, C) TC (A, C, G, T) GC (A, G) AA (A, G, T) AT (A, G) AA (A, G) TC (T, C) TT-3'). They were synthesized with a Biosearch 8700 DNA synthesizer and used without further purification by HPLC or polyacrylamide gel electrophoresis. A 1.7 kb genomic DNA fragment carying the GUKI gene was cloned in pUC-8 in a way SW