( ure 5 shows a plot of mutat...)Randomize Gene Sequences with New PCR Mutagenesis Kit

ure 5 shows a plot of mutation frequency (number of mutations per kb) as a function of target duplication number. As expected, mutation frequency increased with decreasing template concentration (increasing d value). For the lacZ gene, low mutation frequencies (0.08%-0.3%) were produced in PCRs employing 10 ng to 100 ng DNA template (d=5.6-9.4). PCRs performed with 10 pg, 100 pg, or 1 ng of DNA template (d=12.3-19.6) exhibited mutation frequencies ranging from 0.3% to 0.8%, while the highest mutation rates were achieved in reactions employing two (0.7%-1.4%; d=30-34) or three (1.6%; d=49) sequential PCRs.

Fig.5

The GeneMorph kit has been used to mutagenize other target genes, including a 2.5-kb polymerase gene and a 720-bp sequence encoding Green Fluorescent Protein (GFP). (Figure 5)

Mutational Spectrum of Mutazyme

Table 2 shows the types of mutations introduced by Mutazyme DNA polymerase. These data were compiled from DNA sequence analysis (37.5-kb total) of 45 clones randomly selected from lacZ mutagenesis studies. Mutations were distributed uniformly throughout the lacZ gene, rather than localized in hotspots (data not shown). Mutazyme DNA polymerase introduced all possible transition (purinepurine; pyrimidinepyrimidine) and transversion (purinepyrimidine; pyrimidinepurine) mutations, while insertions and deletions were relatively rare (~1%).

Mutational bias is most commonly analyzed by calculating either the ratio of transition (Ts) to transversion (Tv) mutations or the ratio of AT to GC changes (ATGC/GCAT). Since there are eight possible t
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