Random mutagenesis by using mixtures of dNTP and dITP in PCR.

Abstract

Listen

Translate article

Generating random mutations in a DNA fragment of a specific size is often the method of choice for changing specific properties of an encoded protein or for studying the fimctional properties of a specific DNA fragment. In all these cases it is helpful and in some cases essential to have a screening method available that allows for distinction between phenotypic changes brought about by one or more random mutations in the DNA fragment of interest. Several methods have been described using mixed synthetic oligonucleotides for random mutagenesis, which work efficiently but can only be applied for mutagenesis of DNA fragments of limited size (1—3). Alternative methods using polymerase chain reaction (PCR) with reduced fidelity have been described that offer an increased mutation frequency as well as the advantage of random mutations in relatively large DNA fragments and a random distribution of the types of mutation (4—6). However, for many applications it is desirable to use a method that allows the modulation of the desired number of mutations in a DNA fragment of a specific size and the degree of randomness of the mutations. The method described here (7) offers the advantage of choosing preferred nucleotides being randomly incorporated as well as adjusting the mutation frequency to almost any desired level. This method is based on the following principles: