DIRECT RANDOM MUTAGENESIS OF GENE-SIZED DNA FRAGMENTS USING POLYMERASE CHAIN-REACTION

The polymerase chain reaction (PCR) can be used to amplify a DNA fragm ent with the concomitant creation of numerous mutations provided that one dNTP substrate is in excess over the three others. Advantage was t aken of this behavior to systematically mutagenize a 291-bp-long DNA f ragment and to define the rules relating the frequencies of each possi ble bp substitution to the set of the dNTP concentrations in the PCR e xperiment. Sets of parameters governing the rules were determined unde r various mutagenic conditions including the addition of MnC1(2). Fina lly, validity of the rules was assessed in several mutagenesis experim ents showing that a wide range of substitution frequencies including A T --> GC and GC --> AT transitions as well as AT --> TA transversions can be obtained at will. (C) 1995 Academic Press, Inc.