We have previously described the use of mutagenic nucleotide analogues for sequencing small intractable DNA regions (Keith et al., 2004b) (see Figure 1). The nucleotide analogues are not completely random mutagens, rather preferred nucleotide transition reactions are induced (with transversions, insertions and deletions occurring very rarely) in an almost random distribution (Yu et al., 1993; Zaccolo et al, 1996). The different random mutations cause the mutant copies to have different sequences, which are then cloned and sequenced. Mutations introduced at very early rounds of amplification can establish "founder mutations'' that occur in a significant proportion of the progeny amplimers, although these founder mutations are themselves at random loci (Figure 2). These characteristics allow the influence of founder mutations to be minimized through simple experimental design devices. The DNA sequences determined from a low number of the altered copies can then analysed using Bayesian methods to reconstruct the original wild-type sequence (Keith et al., 2003). Surprisingly, this entire process has efficiencies and accuracies roughly equivalent to conventional sequencing.
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