Validation of denaturing high performance liquid chromatography as a rapid detection method for the identification of human INK4A gene mutations
Validation of denaturing high performance liquid chromatography as a rapid detection method for the identification of human INK4A gene mutations
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DOI：
10.1016/s1525-1578(10)60667-8
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发表时间：
2001-11-01
影响因子：
4.1
通讯作者：
Berwick, M
中科院分区：
文献类型：
--
作者：
Orlow, I;Roy, P;Berwick, M
黑色素瘤的发病率在西方国家迅速增加。家族性和一些散发性黑色素瘤的遗传易感性与INK 4A基因突变的存在有关。为了更好地确定基于遗传和环境相互作用的散发性黑色素瘤的风险，需要研究大量病例。缺乏序列变异热点的基因的突变分析是耗时且昂贵的。本文介绍了变性高效液相色谱法（DHPLC）在突变筛查中的应用。从129个样品和13个已知突变体中扩增外显子1 α，2和3，得到347个在不同温度下检查的产物。这些扩增子中的42个在色谱图上显示出明显的非野生型特征。独立测序分析证实了Leu（32）Pro、Ile 49 Thr、88 del G、Gln 50 Arg、Arg 24 Pro、Met 53 Ile、Met 53 Thr、Arg 58 stop、Pro 81 Leu、Asp 84 Ala、Arg 80 stop、Gly 101 Trp、Val 106 Val、Ala 148 Thr和内含子1（-2）位置（C -> T）的16个不同核苷酸变异;和在3' UTR中，核苷酸500（C → G）。DHPLC在突变或多态性的样品中没有得到假阴性或假阳性。我们的结论是DHPLC是一种快速，敏感，成本效益高，可靠的方法，用于扫描INK 4A体细胞或生殖系突变和多态性;大量的样品。
The incidence of melanoma is increasing rapidly in western countries. Genetic predisposition in familial and in some sporadic melanomas has been associated with the presence of INK4A gene mutations. To better define the risk for developing sporadic melanoma based on genetic and environmental Interactions, large groups of cases need to be studied. Mutational analysis of genes lacking hot spots for sequence variations is time consuming and expensive. in this study we present the application of denaturing high performance liquid chromatography (DHPLC) for screening of mutations. Exons 1 alpha, 2, and 3 were amplified from 129 samples and 13 known mutants, yielding 347 products that were examined at different temperatures. Forty-two of these amplicons showed a distinct non-wild-type profile on the chromatogram. independent sequencing analysis confirmed 16 different nucleotide variations in Leu(32)Pro; Ile49Thr; 88 del G; Gln50Arg; Arg24Pro; Met53Ile; Met53Thr; Arg58stop; Pro81Leu; Asp84Ala; Arg80stop; Gly101Trp; Val106Val; Ala148Thr; and in positions (-2) in intron 1 (C --> T); and in the 3' UTR, nucleotide 500 (C --> G). No false negatives or false positives were obtained by DHPLC in samples with mutations or polymorphisms. We conclude that the DHPLC is a fast, sensitive, cost-efficient, and reliable method for the scanning of INK4A somatic or germline mutations and polymorphisms; of large number of samples.