Quick screening of plasmid deletion clones carrying inserts of desired sizes for DNA sequencing

Abstract

Plasmids pWQX001 and pWQX005, constructed from pGEM-4 with an insert of 6.5 kb, were unidirectionally digested with exonuclease III and exonuclease VII. The DNA digests were ligated and used to transform competent cells of Escherichia coli DH5-alpha. The size of the deletion plasmid carried by each transformant was estimated through agarose gel electrophoresis of crude lysates without any purification of the plasmid DNA. Colonies carrying plasmid DNAs with different deletions of the insert were grown and their DNAs were purified through a miniprocedure. The size of each purified plasmid DNA was determined accurately after linearization of the plasmid with an appropriate restriction endonuclease. The remainder of the DNA preparation was sufficiently pure to be sequenced using Sanger's dideoxynucleotide chain termination method. An easy, quick procedure is described for the preliminary selection of templates for DNA sequencing after construction of deletion clones of recombinant plasmid DNA using exonuclease III and exonuclease VII. This procedure permits a rapid screening of large numbers of colonies and selection of those carrying plasmid DNAs with inserts of the desired sizes for sequencing. This procedure does not require purification of the deletion plasmid DNA.