An inexpensive, specific and highly sensitive protocol to detect the BrafV600E mutation in melanoma tumor biopsies and blood.
An inexpensive, specific and highly sensitive protocol to detect the BrafV600E mutation in melanoma tumor biopsies and blood.
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DOI：
10.1097/cmr.0b013e32833d8d48
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发表时间：
2010-10
影响因子：
2.2
通讯作者：
Mier JW
中科院分区：
文献类型：
--
作者：
Panka DJ;Sullivan RJ;Mier JW
BrafV600E 突变已在转移性黑色素瘤、结肠癌、甲状腺癌和其他癌症患者中检测到。最近的研究表明，具有这种突变的肿瘤对 Braf 抑制剂特别敏感——因此需要可靠地确定肿瘤标本的 Braf 状态。目前用于筛选这种突变的技术无法解决与带有野生型 Braf 等位基因的浸润基质和免疫细胞相关的问题，因此可能无法检测突变 BrafV600E 肿瘤的存在。我们开发了一种快速、廉价的方法，可以减少肿瘤活检中野生型 Braf 序列的污染。该方案涉及一系列 PCR 扩增和限制性消化，利用野生型和突变型 Braf RNA 在 600 位的独特特征。使用该方案，可以在含有低至 0.1% BrafV600E 突变细胞的混合群体的 RNA 中检测到突变型 Braf。
The BrafV600E mutation has been detected in patients with metastatic melanoma, colon, thyroid and other cancer. Recent studies suggested that tumors with this mutation are especially sensitive to Braf inhibitors-hence the need to reliably determine the Braf status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild type Braf alleles and thus may fail to detect the presence of mutant BrafV600E tumors. We have developed a rapid, inexpensive method that reduces the contamination of wild type Braf sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild type and mutant Braf RNA at position 600. Using this protocol, mutant Braf can be detected in RNA from mixed populations with as few as 0.1% BrafV600E mutant cells.