Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis.
Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case-control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.
Nguyen-Dumont
Tú
T
International Agency for Research on Cancer (IARC), Lyon, France.
Calvez-Kelm
Florence Le
FL
Forey
Nathalie
N
McKay-Chopin
Sandrine
S
Garritano
Sonia
S
Gioia-Patricola
Lydie
L
De Silva
Deepika
D
Weigel
Ron
R
Sangrajrang
Suleeporn
S
Lesueur
Fabienne
F
Tavtigian
Sean V
SV
D064007:Ataxia Telangiectasia Mutated Proteins; D001943:Breast Neoplasms / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases