High-Throughput Screening for Known Mutations by Automated Analysis of Single Sequencing Reactions

Abstract

This work shows that single sequencing reactions analyzed on an automated DNA sequencer can be an efficient way of screening PCR products for known mutations. We have analyzed a mutation in exon 10 of the human aromatase gene and show that an unambiguous genotype could be elucidated in more than 90% of the analyzed samples. Compared to analysis by full sequencing, 4 times more samples can be analyzed per gel, so that the sample capacity of the gel is approaching that of alternative gel-based methods for genotype analysis. Unlike many of these, the method offers direct identification of the variant sequence position and on-line analysis without the need of post-electrophoretic processing of the gel.