Enzymatic inverse polymerase chain reaction library mutagenesis

Abstract

This invention discloses a method for generating a recombinant library by introducing one or more changes within a predetermined region of double-stranded nucleic acid, comprising providing a first primer population and a second primer population, each of the populations having a variable base composition at known positions along the primers, the primers incorporating a class IIS restriction enzyme recognition sequence, being capable of directing change in the nucleic acid sequence and being substantially complementary to the double stranded nucleic acid to permit hybridization thereto. The method additionally comprises hybridizing the first and second primer populations to opposite strands of the double stranded nucleic acid to form a first pair of primer-templates oriented in opposite directions, performing enzymatic inverse polymerase chain reaction to generate at least one linear copy of the double stranded nucleic acid incorporating the change directed by the primers, cutting the double stranded nucleic acid copy with a class IIS restriction enzyme to form a restricted linear nucleic acid molecule containing the change, joining termini of the restricted linear nucleic acid molecule to produce double-stranded circular nucleic acid and introducing the nucleic acid into compatible host cells. A method is additionally provided for generating a recombinant library using wobble-base mutagenesis.