( Rs carried out with 10 pg to...)Randomize Gene Sequences with New PCR Mutagenesis Kit

Rs carried out with 10 pg to 1 ng of DNA template introduced errors at a rate of three to seven mutations per kb (0.3%-0.7%). Mutation frequencies greater than 0.7% are achieved by performing sequential amplifications. By reamplifying a small portion of a PCR reaction, the number of target doublings can be substantially increased. Mutation frequencies of 0.6% to 1.3% or 0.9% to1.8% were produced by performing two or three sequential PCR reactions, respectively. (Table 1)

Random Mutagenesis of Target Genes

Fig.4

The efficiency and ease of use of the GeneMorph kit was demonstrated in studies employing lacZ as the mutational target gene. A 650-bp DNA fragment containing a portion of the lacZ gene was amplified with Mutazyme DNA polymerase from varying amounts of plasmid DNA (Figure 4). In this study, the PCR primers were designed to introduce LIC (ligation independent cloning) sites for cloning into the Affinity LIC vector. Mutazyme produced high yields of the 650-bp amplicon from 10 pg up to 100 ng of DNA template (Figure 4). Product yields were quantified by comparison to a DNA standard, and total yields ranged from 1 mg to 7.5 mg per 50-ml PCR reaction. In addition, a small portion of one reaction (Figure 4, Lane 5) was reamplified in two or three sequential PCR reactions (Figure 4, Lanes 8 and 9).

The frequency of mutations in the lacZ gene was determined for numerous PCR reactions employing various concentrations of DNA template. PCR products were gel purified and cloned into the Affinity vector (Methods). For each PCR reaction, 2 to 10 clones were randomly selected and sequenced. Fig
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