Division of Pathology, School of Molecular Medical Sciences, University of Nottingham, Nottingham, UK.
Int J Exp Pathol. 2010 Dec;91(6):500-5. doi: 10.1111/j.1365-2613.2010.00733.x. Epub 2010 Sep 7.
Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.
突变检测在癌症管理中很重要。有几种方法可供选择，其中高分辨率熔解（HRM）分析和焦磷酸测序是最通用的。我们对这些技术进行了比较分析。这些方法是：为了比较检测限（LOD），对 KRAS（密码子 12/13 热点）和 BRAF（V600E 热点）的突变进行了测试。分析了含有突变等位基因频率约为 25%/12.5%/6%/3%/1.5%/0.8%的 DNA 混合物。为了比较突变检测频率，对 22 个 DNA 样本（来自细胞系的 9 个高质量样本，来自福尔马林固定石蜡包埋组织的 13 个低质量样本）进行了三个 KRAS（密码子 12/13、61 和 146）和两个 BRAF（V600E 和外显子 11）热点的突变检测。KRAS（密码子 12/13）和 BRAF（V600E）的 HRM 分析表明，分别可以可靠地检测到 3%和 1.5%的突变等位基因，而焦磷酸测序在每种情况下都可以可靠地检测到 6%的突变等位基因。在对 22 个 DNA 样本进行的 110 次测试中，HRM 和焦磷酸测序在 109 次测试中给出了相同的结果。在测试的两个样本中，先前通过直接 Sanger 测序被称为 KRAS 野生型，但通过 HRM 和焦磷酸测序均被发现为突变型。HRM 和焦磷酸测序都可以检测少量的突变等位基因，尽管 HRM 的检测下限较低。这两种方法都适用于突变检测，并且都比 Sanger 测序更敏感。
