荧光染料SYTO13用于高分辨熔解曲线技术SNP基因分型的评价

王倩; 闫雯; 沈明辉; 马克君; 郜莉娜; 尤崇革

doi:10.3760/cma.j.issn.1009-9158.2017.02.004

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中华检验医学杂志

• 论著 •

ENGLISH ABSTRACT

荧光染料SYTO13用于高分辨熔解曲线技术SNP基因分型的评价

作者及单位信息

出版日期： 2017 -02 -11 ·

DOI： 10.3760/cma.j.issn.1009-9158.2017.02.004

Evaluation of SYTO13 as fluorescent dye for high resolution melting based single nucleotide polymorphism genotyping

Authors Info & Affiliations

Wang Qian

First Department of Clinical Laboratory, Lanzhou University Second Hospital, Key Laboratory of Digestive System tumors of Gansu Province, Lanzhou 730030, China

Yan Wen

Shen Minghui

Ma Kejun

Gao Li′na

You Chongge

Published： 2017 -02 -11 ·

DOI： 10.3760/cma.j.issn.1009-9158.2017.02.004

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摘要

目的评价荧光染料SYTO13在高分辨熔解曲线技术(HRM)检测单核苷酸多态性(SNP)位点的基因分型能力和临床适用性。

方法实验性能验证。选择经测序确认rs3125734 C>T(Ⅰ类SNP )、rs255758 A>C(Ⅱ类SNP)和rs688C>T三个位点基因型的样本36例，以商业化HRM染料LCGreen Plus为对照，进行SYTO13对Ⅰ类和Ⅱ类SNP以及rs688C>T位点的长片段扩增(107 bp)与短片段扩增(52 bp)基因分型能力的评价。基因分型能力以野生型与纯合突变型的Tm差值ΔTm表示，独立样本 t检验比较野生型和纯合突变型的Tm均值，两种染料之间检测能力的比较分析采用配对 t检验。临床适用性以随机选取已测序分型的35例样本进行批量均相检测来评价。

结果SYTO13能够清楚直观地对Ⅰ类和Ⅱ类SNP进行基因分型(ΔTm _classⅠ＝0.36±0.05， t _classⅠ＝14.827， P _classⅠ＝0.000；ΔTm _classⅡ＝0.42±0.110， t _classⅡ＝9.539， P _classⅡ＝0.000)。均相检测时SYTO13对Ⅰ类SNP的基因分型直观性更好(ΔTm _SYTO13＝0.39±0.027)，而对短片段扩增产物基因分型效果不佳。临床适用性评价结果显示SYTO13对长片段扩增的Ⅰ类和Ⅱ类SNP除个别离群样本外均能准确基因分型，离群样本经复查后也能准确分型。

结论SYTO13可作为HRM染料准确地对长片段扩增的Ⅰ类和Ⅱ类SNP位点进行基因分型，适合于临床常规应用。(中华检验医学杂志，2017, 40：88－94)

关键词：多态性，单核苷酸;基因分型技术;荧光染料;高分辨率熔解曲线

ABSTRACT

ObjectiveTo evaluate the utility of fluorescent dye SYTO13 for high-resolution melting (HRM) detection in single nucleotide polymorphism (SNP) genotyping and its clinical application.

MethodsThis is a performance verification study. 36 genotype defined samples were divided into three groups: SNP rs3125734 C>T (class Ⅰ SNP) , rs255758 A>C (class ⅡSNP) and rs688C>T. These samples were used to evaluate SYTO13′s SNP genotyping capability of class Ⅰ SNP, class Ⅱ SNP, and two PCR products of different lengths (52 and 107 bp) covering the same SNP of rs688C>T. The commercial HRM dye of LCGreen Plus was used as the control. The genotyping capability is indicated by the Tm difference(ΔTm) between wild type and homozygous mutant genotypes. The Tm differences between wild genotype and homozygous mutant genotype were compared using the Independent Samples t test. Paired t test was used to evaluate genotyping capability of the two dyes. The clinical applicability is evaluated by synchronously performing PCR amplification and HRM analysis on thirty-five randomly selected DNA samples with known genotypes of the three SNPs.

ResultsThe SNPs of class Ⅰ and class Ⅱ can be genotyped directly and clearly with SYTO13 (ΔTm _classⅠ=0.36±0.05, t _classⅠ =14.827, P _classⅠ=0.000; ΔTm _classⅡ=0.42±0.110, t _classⅡ=9.539, P _classⅡ=0.000). The classⅠSNP genotyping results was better using SYTO13 (ΔTm _SYTO13=0.39±0.027), while the SNP genotyping for small amplicon did not discriminated clearly in this study. Long amplicons of class Ⅰ and Ⅱ SNPs can be identified directly except for several samples which can be genotyped accurately after having performed reexamination.

ConclusionSYTO13 can apply for HRM analysis of genotyping classⅠ and Ⅱ SNPs with long amplicon and for clinical routine detection.( Chin J Lab Med, 2017, 40: 88-94)

KEYWORDS： Polymorphism, single nucleotide;Genotyping techniques;Fluorescent dyes;High resolution melting

Corresponding author: You Chongge, Email: nc.defudabe.uzlghcuoy

FUNDING:

National Natural Science Foundation of China(81560343); Science and Technology Management Program of Health Care Industry in Gansu Province(GWGL2014-02); Doctoral Scientific Research Fund of Lanzhou University Second Hospital(Ynbskyjj2015-1-6)

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All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

引用本文

王倩,闫雯,沈明辉,等. 荧光染料SYTO13用于高分辨熔解曲线技术SNP基因分型的评价[J]. 中华检验医学杂志,2017,40(2)：88-94.

DOI:10.3760/cma.j.issn.1009-9158.2017.02.004

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HRM技术是基于嵌入饱和荧光染料的双链DNA在高温变性时产生特征熔解曲线进行基因突变扫描和基因分型的一种新方法 ^{[
1
]}，由于其高效、特异、操作简便、快速、成本低和均相检测的特点而被广泛应用 ^{[
2
,

3
,

4
,

5
,

6
]}。HRM技术实施DNA序列分析检测的两个基本条件是高分辨温度检测的PCR仪器和饱和荧光染料。目前已报道常用的HRM染料有LCGreen Plus、EvaGreen、Resolight和SYTO9 ^{[
7
]}，这些染料相对价格较贵，因此，寻找相对价格低廉的HRM荧光染料有利于HRM技术的推广和普及。SYTO13与SYTO9同属花青素核酸荧光染料家族，但价格不足SYTO9的一半，2007年Gudnason等 ^{[
8
]}首先报道了SYTO13在抑制PCR反应、改变DNA熔解温度和优先结合GC片段三个方面优于SYBR Green，可用于实时荧光定量PCR。2011年Eischeid ^{[
9
]}进一步发现SYTO13的荧光信号强度和PCR扩增效率优于SYBR Green，并与商品化荧光染料EvaGreen进行比较，发现两者相当。2015年Radvanszky等 ^{[
10
]}再次验证SYTO13可用于real－time PCR，并进行了小片段单碱基突变基因( GJB2基因G>A，52 bp)、较大序列变异( DMPK基因 Alu插入/缺失多态性，493 bp/381 bp)、无标记探针的HRM基因分型与突变扫描，得出SYTO13虽不及商品化HRM荧光染料LCGreen Plus和EvaGreen但也可用于HRM检测。尽管该文献探索了SYTO13可否用于HRM检测，但对其HRM检测性能和应用特点未作评价。本研究对SYTO13基因分型Ⅰ类SNP(C/T或G/A)和Ⅱ类SNP(C/A或G/T)以及长片段PCR产物与短片段PCR产物的检测能力和临床适用性进行评价，并总结归纳其应用要点。

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摘要
参考文献

参考文献

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Wittwer CT , Reed GH , Gundry CN ,et al. High－resolution genotyping by amplicon melting analysis using LCGreen[J]. Clin Chem, 2003,49(6Pt1):853-860. DOI: 10.1373/49.6.853 .