Tindall Elizabeth A, Petersen Desiree C, Woodbridge Paula, Schipany Katharina, Hayes Vanessa M
Cancer Genetics Group, Children's Cancer Institute Australia for Medical Research, Sydney Children's Hospital, Randwick, Sydney, NSW, Australia.
Hum Mutat. 2009 Jun;30(6):876-83. doi: 10.1002/humu.20919.
Mutation detection has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to design. Nongel-based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/clinical setting. In this study, we assessed the latest of the nongel-based methods, namely high-resolution melt (HRM) curve analysis. HRM is a closed-tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner System using LC Green PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of double-stranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC-rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis.
直到最近，突变检测还严重依赖基于凝胶的方法，这些方法既耗时又难以设计。因此，非凝胶系统对于提高简便性和缩短周转时间很重要，同时又不影响检测的灵敏度和准确性，尤其是在诊断/临床环境中。在本研究中，我们评估了最新的非凝胶方法，即高分辨率熔解（HRM）曲线分析。HRM是一种闭管方法，在DNA扩增过程中加入一种饱和染料，然后随着DNA双链因温度升高而变性，监测荧光变化。我们使用两个平台评估了来自八个基因（即SERPINA1、CXCR7、MBL、VDR、NKX3A、NPY、TP53和HRAS）的10个扩增子，一个是使用LC Green PLUS DNA结合染料的LightScanner系统（美国犹他州盐湖城的Idaho Technology公司），另一个是使用HRM Master染料的LightCycler 480（美国印第安纳州印第安纳波利斯的罗氏诊断公司）。DNA变异（突变或多态性）先前是使用变性梯度凝胶电泳（DGGE）鉴定的，这是一种与HRM类似的方法，基于双链DNA不同的熔解特性。根据变异和片段复杂性选择片段。这包括多个序列变异的存在、不同方向的变异以及富含GC的片段中的单个或多个变异（构成性或体细胞性）。我们证明了HRM方法在分析复杂DNA区域时目前存在的局限性，并呼吁在将HRM作为基于基因分析进行临床诊断的唯一方法时要谨慎。
