A PCR-derived library of random point mutations within the V3 region of simian immunodeficiency virus

Oligonucleotide primers corresponding to variable region 3 (V3) of simian immunodeficiency virus (SIV) were randomly mutagenized during synthesis by doping each of the four nucleoside phosphoramidites with a small amount of the other three. PCR was then used to incorporate the altered sequences into larger, clonable DNA fragments by spliced overlap extension (SOE). With the composition of the phosphoramidites used, 53 of the 100 clones analyzed were unique, having one or more point mutation within the 84-bp target sequence. These 53 unique clones contained an average of 2.1 nucleotide substitutions and 1.5 amino acid substitutions per clone within the target V3 sequence. Of the internal 25 amino acid positions within the V3 domain, 23 were changed at least once. This method should be generally useful for the construction of libraries of random point mutations within a defined target DNA sequence.