Song Chen, Milbury Coren A, Li Jin, Liu Pingfang, Zhao Meiping, Makrigiorgos Gerasimos Mike
Department of Radiation Oncology, Division of Medical Physics and Biophysics, Harvard Medical School, Boston, MA, USA.
Diagn Mol Pathol. 2011 Jun;20(2):81-9. doi: 10.1097/PDM.0b013e3181fde92f.
KRAS mutations exhibit significant predictive and prognostic value in cancer. Efficient, sensitive, and accurate molecular approaches are required to evaluate KRAS mutation status, even when mutant alleles are restricted to a small portion of a clinical sample, which otherwise contains wild-type alleles. We describe a highly sensitive method to detect KRAS mutations by high-resolution melting (HRM) analysis after mutation enrichment by fast-COLDpolymerase chain reaction (PCR). Using 10 ng of starting DNA and after fast-COLD-PCR of a 76-bp region containing KRAS codons 12 and 13; the amplicons undergo a nested conventional PCR reaction followed by HRM analysis. Samples exhibiting aberrant melting profiles are sequenced to identify mutation type and position. Serial dilution experiments indicate a sensitivity of approximately 0.3% mutant-to-wild type for HRM-based mutation detection and the ability to directly sequence mutation-containing samples. A number of lung adenocarcinoma specimens earlier characterized were screened. Fast-COLD-PCR-HRM analysis correctly identified KRAS mutations and also showed a previously undetected, low-level missense GGT > TTT complex mutation. On account of the short target regions and low requirement of starting DNA, this rapid, cost-effective, and sensitive fast-COLD-PCR-HRM approach is expected to find broad application for detecting low-abundance KRAS mutations in a wide range of clinical specimens.
KRAS突变在癌症中具有显著的预测和预后价值。即便突变等位基因仅占临床样本的一小部分，而样本中其他部分为野生型等位基因，仍需要高效、灵敏且准确的分子方法来评估KRAS突变状态。我们描述了一种高灵敏度的方法，即通过快速COLD聚合酶链反应（PCR）富集突变后，利用高分辨率熔解（HRM）分析来检测KRAS突变。使用10 ng起始DNA，对包含KRAS密码子12和13的76 bp区域进行快速COLD-PCR后，扩增子进行巢式常规PCR反应，随后进行HRM分析。对熔解曲线异常的样本进行测序，以确定突变类型和位置。系列稀释实验表明，基于HRM的突变检测灵敏度约为0.3%突变型对野生型，且能够直接对含突变样本进行测序。对一些先前已鉴定的肺腺癌标本进行了筛查。快速COLD-PCR-HRM分析正确鉴定了KRAS突变，还发现了一个先前未检测到的低水平错义GGT > TTT复合突变。由于靶区域短且对起始DNA要求低，这种快速、经济高效且灵敏的快速COLD-PCR-HRM方法有望在广泛的临床标本中检测低丰度KRAS突变方面得到广泛应用。
