De Leeneer Kim, Coene Ilse, Poppe Bruce, De Paepe Anne, Claes Kathleen
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Clin Chem. 2008 Jun;54(6):982-9. doi: 10.1373/clinchem.2007.098764. Epub 2008 Apr 10.
High-resolution melting is an emerging technique for detection of nucleic acid sequence variations. Developments in instrumentation and saturating intercalating dyes have made accurate high-resolution melting analysis possible and created opportunities to use this technology in diagnostic settings. We evaluated 2 high-resolution melting instruments for screening BRCA1 and BRCA2 mutations.
To cover the complete coding region and splice sites, we designed 112 PCR amplicons (136-435 bp), amplifiable with a single PCR program. LCGreen Plus was used as the intercalating dye. High-resolution melting analysis was performed on the 96-well Lightscanner (Idaho Technology Inc.) and the 96-well LightCycler 480 (Roche) instruments. We evaluated sensitivity by analyzing 212 positive controls scattered over almost all amplicons and specificity by blind screening of 22 patients for BRCA1 and BRCA2. In total, we scanned 3521 fragments.
All 212 known heterozygous sequence variants were detected on the Lightscanner by analysis on normal sensitivity setting. On the LightCycler 480, the standard instrument sensitivity setting of 0.3 had to be increased to 0.7 to detect all variants, decreasing the specificity to 95.9% (vs 98.7% for the Lightscanner).
Previously, we screened BRCA1/2 by direct sequencing of the large exon 11 and denaturing gel gradient electrophoresis (DGGE) for all other coding exons. Since the introduction of high-resolution melting, our turnaround time has been one third of that with direct sequencing and DGGE, as post-PCR handling is no longer required and the software allows fast analyses. High-resolution melting is a rapid, cost-efficient, sensitive method simple enough to be readily implemented in a diagnostic laboratory.
高分辨率熔解分析是一种新兴的核酸序列变异检测技术。仪器设备和饱和嵌入染料的发展使得准确的高分辨率熔解分析成为可能，并为在诊断环境中应用该技术创造了机会。我们评估了2种高分辨率熔解仪器用于筛查BRCA1和BRCA2突变。
为覆盖完整的编码区和剪接位点，我们设计了112个PCR扩增子（136 - 435 bp），可通过单一PCR程序进行扩增。使用LCGreen Plus作为嵌入染料。在96孔Lightscanner（爱达荷技术公司）和96孔LightCycler 480（罗氏公司）仪器上进行高分辨率熔解分析。我们通过分析分布在几乎所有扩增子上的212个阳性对照评估敏感性，并通过对22例患者进行BRCA1和BRCA2的盲筛评估特异性。总共，我们扫描了3521个片段。
在Lightscanner上，通过正常灵敏度设置分析检测到了所有212个已知的杂合序列变异。在LightCycler 480上，标准仪器灵敏度设置0.3必须提高到0.7才能检测到所有变异，特异性降至95.9%（而Lightscanner为98.7%）。
以前，我们通过对大的第11外显子进行直接测序以及对所有其他编码外显子进行变性凝胶梯度电泳（DGGE）来筛查BRCA1/2。自从引入高分辨率熔解分析以来，我们的周转时间是直接测序和DGGE的三分之一，因为不再需要PCR后处理，并且软件允许快速分析。高分辨率熔解分析是一种快速、经济高效、灵敏的方法，简单到足以在诊断实验室中轻松实施。
