A RAPID SINGLE-STRANDED CLONING STRATEGY FOR PRODUCING A SEQUENTIAL SERIES OF OVERLAPPING CLONES FOR USE IN DNA SEQUENCING - APPLICATION TO SEQUENCING THE CORN MITOCHONDRIAL 18-S RDNA

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary [c] DNA oligomers to form specific cleavage and ligation substrates. It was independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3'' to 5'' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on an agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in 1 tube in < 1 day. The procedure was used to subclone and sequence the maize mitochondrial 18 S r[ribosomal]DNA and 5'' flanking region (2622 bases) in < 1 wk. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions and cDNAs are discussed.