High-resolution genotyping by amplicon melting analysis using LCGreen
High-resolution genotyping by amplicon melting analysis using LCGreen
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DOI：
10.1373/49.6.853
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发表时间：
2003-06-01
影响因子：
9.3
通讯作者：
Pryor, RJ
中科院分区：
文献类型：
--
作者：
Wittwer, CT;Reed, GH;Pryor, RJ
背景资料：最近引入了高分辨率扩增子解链分析作为用于基因分型和突变扫描的闭管方法（Gundry等人，Clin Chem 2003;49：396-406）。该技术需要荧光标记的引物，并且限于检测位于标记引物的解链结构域中的突变。我们的目的是开发一个封闭的基因分型和突变扫描系统，不需要标记oligonucleotides.Methods：我们研究了羟色胺受体2A（HTR 2A）基因（T102 C），β-珠蛋白（血红蛋白S和C）基因，囊性纤维化（F508 del，F508 C，I507 del）基因的多态性。在双链DNA染料LCGreen的存在下进行PCR，并获得高分辨率的扩增子熔解曲线。在荧光归一化、温度调节和/或差异分析后，通过曲线形状和/或位置来区分序列改变。结果：在110 bp的扩增子中，6种常见的β-珠蛋白基因型（AA、AS、AC、SS、CC和SC）均被区分开来。HTR 2A单核苷酸多态性基因分型在一个544 bp的片段，分为两个解链域。由于熔解曲线采集仅需1-2 min，扩增和分析在10-20 min内实现快速循环conditions.Conclusions：高分辨率熔解分析的PCR产物扩增在LCGreen的存在下，可以识别杂合和纯合序列变异。该技术只需要通常的未标记的引物和一个通用的双链DNA染料添加前PCR扩增子基因分型，是一个有前途的方法突变筛查。(C)2003年美国临床化学协会。
Background: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides.Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR.Results: The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions.Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening. (C) 2003 American Association for Clinical Chemistry.