高分辨熔解技术常用荧光染料的SNP基因分型能力比较  

Comparison of common fluorescent dyes for SNP genotyping in HRM technology

作　　者：尤崇革[1,2] 李晓军[1] 郜莉娜[2] 李菲菲[2] 

机构地区：[1]南京军区南京总医院解放军临床检验医学研究所,南京210002 [2]兰州大学第二医院中心实验室,兰州730030

出　　处：《临床检验杂志》2011年第8期575-577,共3页Chinese Journal of Clinical Laboratory Science

基　　金：中国博士后科学基金资助项目(20080430239)

摘　　要：目的评价LC Green、Syto 9和Eva Green 3种高分辨熔解(HRM)染料对单核苷酸多态性(SNP)基因分型的能力。方法以肿瘤坏死因子(TNF)基因启动子-857 C>T(rs1799724)位点为例,分别用上述3种荧光染料进行PCR-HRM检测,依据特征熔解曲线进行基因分型并测序验证。基因分型能力评价以野生型与纯合突变型PCR产物Tm值之差(△Tm)来表示。结果 PCR-HRM检测到4种特征熔解曲线,经测序后发现第4条为双突变杂合,包含-863C>A(rs1800630)位点。HRM染料LC Green,Syto 9,EVa Green的基因分型能力分别为(0.52±0.030)℃、(0.51±0.066)℃和(0.39±0.152)℃。其中Syto 9野生型(CC)和突变型(TT)的变异系数(CV)均为0.03%,为3种染料中最低。结论 3种染料均能用于HRM技术进行SNP基因分型,Syto 9和LC Green分辨率优于Eva Green,其中Syto 9易用性最好、Eva Green性价比最高。Objective To evaluate the capability of three high-resolution melting（HRM） dyes including LC Green,Syto 9 and Eva Green applied in HRM technology for single nucleotide polymorphism（SNP） genotyping.Methods TNF promoter-857CT（rs1799724） was selected to evaluate the above three fluorescent dyes and their capabilities for SNP genotyping in HRM analysis were determined on the basis of the differences of Tm value of PCR products between wild type and homozygous mutant type.Results Four kinds of characteristic HRM curves were observed and the PCR products of the four melting curves were sequenced.The fourth melting curve was verified to be a dual-heterozygote which contained another SNP of TNF promoter-863CA（rs1800630） besides-857CT.The Tm value of resolution for SNP genotyping by LC Green,Syto 9 and Eva Green dyes were（0.52±0.030） ℃,（0.51±0.066） ℃ and（0.39±0.152） ℃ respectively.In the three HRM dyes,Syto 9 showed the lowest coefficients of variation 0.03% in both wildtype and mutant type.Conclusion All the three fluorescent dyes can be applied to HRM technology for SNP genotyping.Syto 9 and LC Green are better than Eva Green in detecting resolution while Syto 9 is the best ease-of-use dye and Eva Green is the highest cost-effective dye.

关 键 词：LC Green Syto 9 EVA Green 高分辨熔解技术 单核苷酸多态性 

分 类 号：Q75[生物学—分子生物学]