Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations
Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations
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DOI：
10.1002/humu.10097
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发表时间：
2002-01-01
期刊：
影响因子：
3.9
通讯作者：
Whittemore, AS
中科院分区：
文献类型：
--
作者：
Andrulis, IL;Anton-Culver, H;Whittemore, AS
BRCA 1是一个大型多外显子基因，有许多方法用于突变分析。比较了五种检测产生提前终止密码子的突变的方法，预测这些突变会导致BRCA 1中截短蛋白的合成。这些包括四种基于DNA的方法：二维基因扫描（TDGS），变性高效液相色谱（DHPLC），酶突变检测（EMD），单链构象多态性分析（SSCP）和基于RNA/DNA的蛋白质截短试验（PTT），有和没有互补5'测序。检测了从21个编码的淋巴母细胞样细胞系样品中分离的DNA和RNA样品。这些标本先前已通过直接自动DNA测序进行了分析，这被认为是突变检测的最佳方法。该组21个细胞系包括14个具有13个独特移码或无义突变的样品，3个具有2个独特剪接位点突变的样品，和4个没有有害突变的样品。本研究的重点是蛋白质截短突变的检测，这些突变最常被报道为与家族中的癌症分离的致病改变。用互补5'测序的PTT正确鉴定了所有15种有害突变。毫不奇怪，基于DNA的技术没有检测到外显子22的缺失。EMD和DHPLC鉴定了除22号外显子缺失外的所有突变。TDGS最初遗漏了两个突变，但在测试设计的轻微变化后可以检测到，SSCP遗漏了五个截短突变。使用互补方法进行突变分析仍然很重要。
A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP It will continue to be important to use complementary methods for mutational analysis.