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Randomize Gene Sequences with New PCR Mutagenesis Kit

Fig.2

Mutation frequency (mutations/kb) is the product of DNA polymerase error rate and number of target duplications (Figure 2). In the GeneMorph kit, a sufficiently high error rate is achieved through use of Mutazyme DNA polymerase. Low, medium, or high mutation frequencies are obtained simply by varying the initial target DNA concentration in the PCR reaction. For the same PCR yield, targets amplified from low amounts of target DNA undergo more duplications than targets amplified from high concentrations of DNA. This principle is illustrated in Figure 3, which shows the number of times an amplicon is copied to produce theoretical yields of 0.5 mg or 10 mg. The more times a target is replicated, the more errors accumulate. Therefore, higher mutation frequencies are achieved simply by lowering input DNA template concentration. Conversely, lower PCR mutation frequencies can be achieved by using higher DNA template concentrations or fewer PCR cycles to limit the number of target duplications.

Fig.3

Selecting the Mutation Frequency of GeneMorph Reactions

Table I presents the initial amount of target DNA required to produce the desired mutation frequency using the GeneMorph kit. For simplicity, we divided the range of mutation frequencies produced by Mutazyme DNA polymerase into three different levels: low (0-3 mutations/kb), medium (3-8 mutations/kb), and high (>8 mutations/kb). Reactions employing 10 ng to 100 ng of target DNA produced mutation frequencies up to three mutations per kb (0.3%), while PC
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