A simple nonisotopic method for restriction mapping in single-stranded DNA cloning vectors based on taking timepoints during primed Klenow synthesis.

Abstract

A fast, simple, and nonisotopic method for restriction mapping inserts in single-stranded cloning vectors (such as M13 or single-stranded plasmids) is presented. The procedure uses a commercially available oligonucleotide sequencing primer to initiate Klenow-mediated, unidirectional DNA synthesis along the single-stranded insert DNA. Aliquots taken at very short timepoints from this reaction are quick-frozen, heat-inactivated, and restriction-digested with the restriction enzyme or enzymes of interest. When the samples are run on an agarose gel and stained with ethidium bromide, the restriction bands appear in the order of their proximity to the priming site. The method's advantages are that it is fast, unidirectional and thus relatively unambiguous, requires neither isotope nor elaborate DNA handling or extraction procedures, and resolves the ambiguities due to "near doublets" that often plaque double-digest mapping and partial-digest mapping. Tetranucleotide restriction maps extending up to 5 kb can be determined from a single priming experiment; more infrequent hexanucleotide restriction sites can be mapped over longer distances. Also, a single aliquot taken at an early timepoint can be restriction-digested to establish the orientation of cloned inserts.