High-resolution melt curve analysis as a high-throughput screening method of large genes

Abstract

High-resolution melt curve analysis (HRM) has been recently introduced as a promising technique for genotyping and mutation scanning in diagnostics. To screen the full coding regions and splice junction sites of BRCA1 and BRCA2 genes, amplification and HRM of 90 PCR amplicons were performed on the LightScanner-32™ (BioFire Diagnostics Inc.) instrument. Sensitivity of the method was evaluated by analyzing 82 clinically damaging mutations distributed in different amplicons and specificity by blind screening of 15 patients for BRCA1 and BRCA2. All known heterozygous variants were detected on the LightScanner-32 by analysis on normal sensitivity setting. 7 DNA-sequence variants had been detected among 15 patients, which all were confirmed by Sanger sequencing. Therefore, HRM is a cost-efficient, sensitive method suitable for high-throughput mutation screening for diagnostic purposes, particularly for large genes such as BRCA1 and BRCA2.