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 DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing 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. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy 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 complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.
Fleming
N
N
Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, CF14 4XN, Cardiff, UK.
Maynard
J
J
Tzitzis
L
L
Sampson
J R
JR
Cheadle
J P
JP
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins
D017931:DNA Primers; D012097:Repressor Proteins; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins