Gundry Cameron N, Vandersteen Joshua G, Reed Gudrun H, Pryor Robert J, Chen Jian, Wittwer Carl T
Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132, USA.
Clin Chem. 2003 Mar;49(3):396-406. doi: 10.1373/49.3.396.
Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR.
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 approximately 1 min and no sample processing was needed after PCR.
Polymorphisms in the HTR2A (T102C), beta-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 degrees C/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4 degrees C/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 <0.2 degrees C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5' tail of identical sequence was added to one of the two unlabeled primers.
High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.
用于鉴定DNA序列变异体的常用方法是在PCR后使用凝胶电泳或柱分离。
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。其中一条PCR引物用荧光标记。PCR后，通过高分辨率熔解分析监测扩增子的熔解转变。不同的纯合子通过扩增子熔解温度（T(m)）来区分。杂合子通过异源双链体的低温熔解来鉴定，这会使整体熔解转变变宽。在这两种情况下，熔解分析大约需要1分钟，PCR后无需样品处理。
分析了5-羟色胺受体2A（T102C）、β-珠蛋白[血红蛋白（Hb）S、C和E]以及囊性纤维化（F508del、F508C、I507del、I506V）基因中的多态性。通过对变性产物进行快速冷却（>2℃/秒），随后在熔解分析期间进行快速加热（0.2-0.4℃/秒），能最佳地检测由杂合DNA扩增产生的异源双链体。杂合子通过更宽地熔解转变与纯合子区分开来，并且每个杂合子都有独特形状的荧光熔解曲线。所有测试的纯合子都能相互区分，包括Hb AA和Hb SS，它们的T(m)相差<0.2℃。扩增子长度在44至304 bp之间变化。如果在两条未标记引物之一上添加相同序列的5'尾，则可以使用通用荧光寡核苷酸代替一条标记引物和一条未标记引物。
用标记引物扩增的PCR产物的高分辨率熔解分析可鉴定杂合和纯合序列变异体。
