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Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening.

A crucial factor in the success of positional cloning efforts is the ability to screen rapidly many different candidate genes for mutations. By modifying standard software, we have improved the detection of heterozygous base positions in PCR products sequenced by cycle sequencing. A key element of the method is the incorporation of a modified heterozygote detection algorithm that permits the use of DNA sequence data derived from PCR and sequencing reactions that have not been fully optimized. This allows sequencing runs of average quality to be used. We demonstrate that the sensitivity and specificity of the method are well suited to mutation detection applications such as positional cloning.