IMPROVED METHOD FOR ORDERED DELETIONS FROM A FIXED POINT IN A CLONED INSERT

摘要

An improved method for generating ordered deletions from any fixed point in a cloned insert. Starting with a single-stranded phagemid template, DNA polymerase is used to extend an annealed primer. For DNA sequencing, the polymerase primer is typically selected to hybridize to the vector molecule near the 5' side of the cloned insert. For in vitro mutagenesis from any point in the cloned insert, a custom polymerase primer is constructed to hybridize to the cloned insert just 5' to the desired deletion site. Extention of the primer 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 times aliquots and digestion with a single-strand-specific endonuclease leads to a series of linear nested fragments of the insert, having a common end corresponding to the 5' end of the primer. These molecules contain all of the vector DNA and so are readily circularized and used to transform cells, providing large numbers of deletion clones with targeted breakpoints.