Simulating pseudogene evolution in vitro: Determining the true number of mutations in a lineage

Hypermutagenic PCR has been used to simulate pseudogene evolution of the Es cherichia coli R67 dihydrofolate reductase gene. Each time the most diverge nt clone was used as template for another round of hypermutagenesis. After six rounds, with an average mutation rate of 0.05 per base per round, up to a 46% nucleic acid sequence variation was achieved. For a few clones the p rotein information content could be annihilated. As the intermediates were cloned and sequenced, it was possible to establish the real lineage and com pute the true number of mutations. Not surprisingly the true number of forw ard and back mutations as well as variable sites exceeded those based on co mparing any single intermediate to the initial sequence. However, the true number of forward and backward mutations, as well as the number of variable sites, increased linearly with sequence divergence from the original seque nce, suggesting an empirical means to correct for branch lengths.