A non-radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations
A non-radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations
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DOI：
10.1002/humu.10024
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发表时间：
2002-01-01
期刊：
影响因子：
3.9
通讯作者：
Müller, O
中科院分区：
文献类型：
--
作者：
Kahmann, S;Herter, P;Müller, O
描述了一种新的突变检测方法，这是蛋白质截断法的技术进步。这项新技术是非放射性的，对几乎所有序列突变的检测都非常敏感，这些突变会导致停止信号或翻译框架的移动。该方法包括四个步骤：1)通过结合固定的互补序列从样品中捕获感兴趣的序列拷贝；2)用编码氨基酸的引物对待分析的基因片段进行PCR扩增。3)体外转录和翻译，4)Western blot分析翻译产物。作为对新方法的评估，我们以1比40的稀释率检测突变基因拷贝，与非突变基因相比。使用该方法，我们能够检测结肠直肠癌患者粪便样本中大肠腺瘤性息肉病肿瘤抑制基因（APC）的突变。该突变无法通过对扩增的APC基因片段直接测序检测到。[j] .生物工程学报，2002。(C) 2002 Wiley-Liss, Inc。
A new method for mutation detection is described, which is a technical advancement of the protein truncation test. The new technique is non-radioactive and highly sensitive for detection of virtually all sequence mutations, which lead to a stop signal or to the shift of the translation frame. The method includes four steps: 1) capture of the interesting sequence copies out of the sample by binding to an immobilized complementary sequence, 2) PCR amplification of the gene fragment to be analyzed with primers coding both for amino. and carboxy-terminal tags, 3) in vitro transcription and translation, and 4) analysis of the translation products by Western blot. As an evaluation of the new method, we detected mutated gene copies at a dilution of 1 to 40 compared to the non,mutated gene. Using the method, we were able to detect a mutation in the adenomatous polyposis coli tumor suppressor gene (APC) in a stool sample of a colorectal cancer patient. This mutation could not be detected by direct sequencing of the amplified APC gene fragment. Hum Mutat 19:165-172, 2002. (C) 2002 Wiley-Liss, Inc.