Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction

Abstract

The polymerase chain reaction (PCR) can be used to amplify a DNA fragment with the concomitant creation of numerous mutations provided that one dNTP substrate is in excess over the three others. Advantage was taken of this behavior to systematically mutagenize a 291-bp-long DNA fragment and to define the rules relating the frequencies of each possible bp substitution to the set of the dNTP concentrations in the PCR experiment. Sets of parameters governing the rules were determined under various mutagenic conditions including the addition of MnCl2. Finally, validity of the rules was assessed in several mutagenesis experiments showing that a wide range of substitution frequencies including AT-->GC and GC-->AT transitions as well as AT-->TA transversions can be obtained at will.