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 Escherichia coli R67 dihydrofolate reductase gene. Each time the most divergent 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 protein information content could be annihilated. As the intermediates were cloned and sequenced, it was possible to establish the real lineage and compute the true number of mutations. Not surprisingly the true number of forward and back mutations as well as variable sites exceeded those based on comparing 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 sequence, suggesting an empirical means to correct for branch lengths.