ArticleThe Journal of molecular diagnostics : JMD2009
Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden.
Article in The Journal of molecular diagnostics : JMD, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Immunohistochemical Detection of the Expressed BRCA1 and BRCA2 Proteins in Microenvironment of Malignant Breast Cancerous Tissues Infected with Human Mammary Tumor Virus.Asian Pacific journal of cancer prevention : APJCP · 2023Article
- Efficient identification of SNPs in pooled DNA samples using a dual mononucleotide addition-based sequencing method.Molecular genetics and genomics : MGG · 2017Article
- Monoallelic characteristic-bearing heterozygous L1053X in BRCA2 gene among Sudanese women with breast cancer.BMC medical genetics · 2017Article
- Detection of Two Drug-Resistance Mutants of the Cytomegalovirus by High-Resolution Melting Analysis.Journal of clinical laboratory analysis · 2016Article
- Screening of the BRCA1 gene in Brazilian patients with breast and/or ovarian cancer via high-resolution melting reaction analysis.Familial cancer · 2016Article
- Screening of exon 11 of BRCA1 gene using the high resolution melting approach for diagnosis in Moroccan breast cancer patients.BMC cancer · 2015Article
- BRCA1/2 mutation screening in high-risk breast/ovarian cancer families and sporadic cancer patient surveilling for hidden high-risk families.BMC medical genetics · 2013Article
- Full-exon pyrosequencing screening of BRCA germline mutations in Mexican women with inherited breast and ovarian cancer.PloS one · 2012Article
- Mutation analysis of SLC26A4 for Pendred syndrome and nonsyndromic hearing loss by high-resolution melting.The Journal of molecular diagnostics : JMD · 2011Article
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- Practical tools to implement massive parallel pyrosequencing of PCR products in next generation molecular diagnostics.PloS one · 2011Article
- Detection of the JAK2 mutation in myeloproliferative neoplasms by asymmetric PCR with unlabeled probe and high-resolution melt analysis.Journal of clinical laboratory analysis · 2011Article
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5 authors.
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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.
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