LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures

A number of techniques have been developed as primary screens to scan for D NA sequence variants, including denaturing gradient gel electrophoresis, de naturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. W e have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutation s in the similar to1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed ea sy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally comp lex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previou sly characterised mutations were successfully identified using seven overla pping bi-directional sequencing reactions. Our approach of long-read sequen cing of long-distance PCR products may allow rapid sequencing of multiple e xons of compact genes and may be appropriate as a highly sensitive primary screen for mutations. (C) 2001 Elsevier Science B.V. All rights reserved.