Generation of unidirectional deletion mutants

Abstract

A novel method that allows introduction of unidirectional deletions into cloned DNA is described. This method is based on the use of a mixture of oligodeoxynucleotide primers that have fixed 5' (or 3') ends defining the end point of the deletion and variable 3' (or 5') ends composed of mixtures of all four nucleotides at six positions. The 5' ends of the oligodeoxynucleotides are hybridized to a fixed location of the M13K11RX templates and the 3' ends are hybridized randomly to the DNA to be analyzed. Such oligodeoxynucleotide primers when extended with DNA polymerase can direct deletions of intervening parts of the single-stranded DNA that by design contains multiple Eco K sites; the deletion products are selected on a host strain with the Eco K restriction system (e.g., using JM101 cells). This method is an efficient way of generating a nested set of deletion mutants useful for dideoxy-sequencing. It can also be used for creating a set of deletion mutants with a particular codon at the 5' or 3' end point.