title: Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden.
abstract: We previously validated mutation scanning for BRCA1 and 2 using high-resolution melting curve analysis (HRMCA). Due to recurrent single nucleotide polymorphisms (SNPs), a considerable amount of sequencing work remains after HRMCA, as melting curves for SNPs and deleterious mutations may be similar. Here, we present a simple approach for the optimization of SNP genotyping with HRMCA using unlabeled probes. Protocols were optimized for 14 frequent SNPs in BRCA1 and 2. Two probes contained an additional mismatch to detect a rare polymorphism a few nucleotides upstream. PCR was performed in the presence of LCgreenPlus and analyzed on a Lightscanner. Genotyping assays were optimized with five wild-type, heterozygous, and homozygous mutant samples. Sensitivity and specificity of the assays were evaluated with a blind screening of 95 samples. All unlabeled probes correctly genotyped the SNPs. A 1:5 asymmetric primer ratio produced sufficient probe-strand duplexes to accurately genotype the SNP of interest. The most important parameter to optimize was the number of PCR cycles. By complementing our BRCA1/2 HRMCA with 14 unlabeled probe assays, we reduced the sequencing burden by three-fold. Our simple approach for optimization can be used as a blueprint to design genotyping assays for other genes. This is one of the largest studies reported to date and the first that presents an approach combining genotyping and mutation scanning of two large polymorphic genes.
authors: Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium.
authors: Kim
authors: K
authors: De Leeneer
authors: Ilse
authors: I
authors: Coene
authors: Bruce
authors: B
authors: Poppe
authors: Anne
authors: A
authors: De Paepe
authors: Kathleen
authors: K
authors: Claes
mesh_terms: D051017:Apoptosis Regulatory Proteins; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005838:Genotype; D006801:Humans; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D051017:Apoptosis Regulatory Proteins; C118024:BLID protein, human; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D015345:Oligonucleotide Probes