Amplicon melting analysis with labeled primers: A closed-tube method for differentiating homozygotes and heterozygotes
Amplicon melting analysis with labeled primers: A closed-tube method for differentiating homozygotes and heterozygotes
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DOI：
10.1373/49.3.396
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发表时间：
2003-03-01
影响因子：
9.3
通讯作者：
Wittwer, CT
中科院分区：
文献类型：
--
作者：
Gundry, CN;Vandersteen, JG;Wittwer, CT
背景资料：DNA序列变异体的鉴定常用的方法是PCR后凝胶电泳或柱分离。方法：我们开发了一种仅需PCR和扩增子熔解分析的序列变异体分析方法。PCR引物之一被荧光标记。PCR后，通过高分辨率解链分析监测扩增子的解链转变。通过扩增子熔解温度（Tm）区分不同的纯合子。通过异源双链体的低温解链来鉴定杂合子，这拓宽了整体解链转变。结果：分析了HTR 2A（T102 C）、β-珠蛋白-[血红蛋白（Hb）S、C和E]和囊性纤维化（F508 del、F508 C、1507 del、I506 V）基因的多态性。通过扩增杂合DNA产生的杂合双链体最好通过快速冷却（>2 ℃/s）变性产物，然后在解链分析期间快速加热（0.2-0.4 ℃/s）来检测。杂合子与纯合子的区别在于更宽的熔解过渡，并且每个杂合子具有独特形状的荧光熔解曲线。所有测试的纯合子彼此区分，包括Hb AA和Hb SS，其T-m的差异为：
Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR.Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (T-m). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required similar to1 min and no sample processing was needed after PCR.Results: Polymorphisms in the HTR2A (T102C), beta-globin-[hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, 1507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 degreesC/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4 degreesC/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in T-m by