单核苷酸多态性的碱基淬灭探针技术检测方法的建立  

A novel base-quenched probe technique for detecting single-nucleotide polymorphisms

作　　者：张俊[1] 罗光华[1] 郑璐[1] 张晓膺[2] 徐宁[3] 

机构地区：[1]苏州大学附属第三医院综合实验室,常州213003 [2]苏州大学附属第三医院胸外科,常州213003 [3]瑞典隆德大学临床化学系

出　　处：《中华检验医学杂志》2009年第9期1064-1068,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（30570752）;常州市831工程资助项目（2006.32）;常州市卫生局重大招标项目资助课题（ZD200709）;常州市卫生局资助项目（047182）

摘　　要：目的研发建立1种成本低、效率高、操作简便的单核苷酸多态性（SNP）检测技术。方法参照待测SNP所在DNA序列，设计并合成引物及探针。探针的一端标记6-羧基荧光素，依据碱基淬灭荧光的原理，应用PCR技术结合熔解曲线的方法进行SNP分型。同时通过建立检测载脂蛋白M（apolipoprotein M，apoM）基因T-778C多态性位点（rs805296）、磷脂酶A2（PLA2G7，rs1051931和rs1805017）、单核细胞趋化蛋白（MCPI，rs1024611）和L-ficolin（rs3124953、ss32469537、ss32469544和m7851696）等8个多态性位点的方法阐明碱基淬灭探针技术的工作原理；并用DNA测序技术鉴定其准确性。结果8个SNP均只需1对引物和1个单荧光基团标记的探针即可成功进行基因分型，整个实验可在60-90min内完成检测；目的基因不同等位基因的纯合子在2个不同的熔解温度出现熔解曲线谷。4种碱基淬灭探针中G．淬灭探针的最大荧光增加速度[Max（dF／dT）]约为A、T、C淬灭探针的1／2（t=7．569、7．726、10．36，P〈0．01）；一致性Kappa检验显示，4种碱基淬灭探针检测SNP方法与DNA测序技术的检测结果一致（Kappa=1；P=0．00）。结论碱基淬灭探针检测SNP技术简单、快速、准确，适用于大批量基因分型研究。Objective To develop a novel, simple and cost-effective method for detecting known single-nucleotide polgmorphisms （SNP） genotyping by using base-quenched probes. Methods Primers and probes were designed according to target DNA sequence. 6-carboxyfluorescein （FAM） was directly conjugated to a base at the either 5＇ or 3＇ end of probes. During the melting of the final PCR product, the sequence alteration was detected as a change in the melting temperature of the base-quenched probe. Apolipoprotein M （apoM） SNP T-778C （rs805296） as a target gene was chose to explain the method in detail and to detect other 7 different SNPs including phospholipase A2, group VII （PLA2G7） gene （rs1051931 and rs1805017）, monoeyte chemoattractant protein-1 （MCP1） gene （rs1024611） and L-fieolin gene （rs3124953, ss32469537, rs32469544 and rs7851696） , and used DNA sequencing analysis to identify its accuracy. Results Base-quenched probe technique requires only a pair of primers and one fluorescent probe connected with FAM, and the whole detection procedure of the assay could be fulfilled within 60 to 90 rain. The maximum speed of fluorescence increase [ Max （dF/dT）1, by using G-quenched probe method, was about 2-fold lower than the others （t = 7. 569, 7. 726, 10. 36, P 〈 0. 01 ）. DNA sequencing analysis validated that all four-type base-quenched probes could provide unbiased genotyping results （Kappa = 1, P = 0. 00 ）, although. Conclusion This method is simple, economic and suitable for large-scale genotyping studies.

关 键 词：多态性 单核苷酸 基因型 核酸探针 

分 类 号：R686[医药卫生—骨科学]