Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates.
A simple method is described for generating nested deletions from any fixed point in a cloned inset. Starting with a single-stranded phagemid template, T4 DNA 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 single-stranded template and ending with transformation; no extractions, precipitations or centrifugations are needed. Results are comparable to those obtained with standard Exonuclease III-generated deletion protocols, but there is no requirement for restriction endonuclease digestion or for highly purified double-stranded DNA starting material. This procedure provides a strategy for obtaining nested deletions in either direction both for DNA sequencing and for functional analysis.
Henikoff
S
S
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004270:DNA, Circular / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D013604:T-Phages / Q000201:enzymology; D013698:Templates, Genetic
D004270:DNA, Circular; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III