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Nucleic Acids Research, 1990, Vol. 18, No. 10 2961
© 1990

MOLECULAR BIOLOGY

Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease gapping of circular templates

Steaen Henikoff

Fred Hutchinson Cancer Research Centre Seattle, WA 98104, USA

Received December 28, 1989. Accepted February 20, 1990.

A simple method is described for generating nested deletions from any fixed point in a cloned insert. Starting with a single-stranded phagemid template, T4DNA polymerase is used to extend an annealed primer. This leads to a fully double-stranded circular molecule with a nick or small gap just 5' to the primer. Exonuclease III initiates progressive digestion from the resulting 3' end. Removal of timed aliquots and digestion with a single-strand specific endonuclease leads to a series of linear nested fragments having a common end corresponding to the 5' end of the primer. These molecules are circularized and used to transform cells, providing large numbers of deletion clones with targeted breakpoints. The 6-step procedure involves successive additions to tubes, beginning with a singlestranded template and ending with transformation; no extractions, precipitations or centrifugations are needed. Results are comparable to those obtained with standard Exonuclease Ill-generated deletion protocols, but there is no requirement for restriction endonuclease digestion or for highly purified doublestranded DNA starting material. This procedure provides a strategy for obtaining nested deletions in either direction both for DNA sequencing and for functional analysis.


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