基于分子信标技术的CYP2C8基因139A/G单核苷酸多态性检测的初步研究  

Detection of CYP2C8 gene 139A / G single nucleotide polymorphism based on molecular beacon technology

作　　者：郭美[1] 雷洁[1] 杨昭[1] 陈庆海[1] 

机构地区：[1]第三军医大学西南医院检验科,重庆400038

出　　处：《第三军医大学学报》2015年第11期1075-1079,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81371641;81171419);国家人社部留学人员科技活动择优项目(2013)~~

摘　　要：目的基于分子信标技术设计一种新型的断接式发夹探针,利用该探针建立并优化检测CYP2C8基因139A/G单核苷酸多态性(single nucleotide polymorphism,SNP)的方法。方法首先运用生物信息学软件设计断接式发夹探针,合成目的探针和靶序列,再探索反应体系的最适温度和最适探针浓度比,最后利用该方法对CYP2C8 139A/G的SNP进行检测。结果断接式发夹探针识别靶序列中SNP位点的最适杂交温度为40℃,反应体系中荧光探针与淬灭探针的最适浓度比为1∶4。在最适温度和最适探针浓度比的反应条件下,断接式发夹探针与CYP2C8基因原始靶序列反应体系的荧光强度为(7 632.0±8.7)RFU,而与突变靶序列反应体系的荧光强度为(6 425.7±6.1)RFU,两者差异有统计学意义(P<0.05)。结论新型的断接式发夹探针可以有效区分原始序列和突变序列,是一种可以用于检测CYP2C8基因139A/G SNP位点的新方法。Objective To design a new fracture-connective hairpin probe based on the technique of molecular beacons, and then to establish and optimize a new method to detect the 139A/G single nucleotide polymorphism （SNP） of CYP2C8 gene by the probe. Methods Firstly, the fracture-connective hairpin probe was designed through the bioinformatic software, and then the desired probes and target sequences were synthesized. The optimal temperature and concentration ratio of probe were explored for the reaction system. At last, this method was performed to detect the 139A/G SNP of CYP2C8 gene. Results The optimal hybridization temperature for the fracture-connective hairpin probe was 40 ℃ to distinguish the SNP of target sequence, and the optimal hybridization ratio was 1 ： 4 of fluorescence probe to quenching probe. Under the condition of optimal temperature and ratio, the fluorescent intensity of the reaction system of the fracture- connective hairpin probe and CYP2C8 gene primary sequence was 7 632.0 ± 8.7 RFU, while that of of the fracture-connective hairpin probe and CYP2C8 gene mutational sequence was 6 425.7 ± 6. 1 RFU. There was difference significance between the 2 reaction systems （ P 〈 0.05 ）. Conclusion Our new type of fracture- connective hairpin probe can effectively distinguish the primary and mutational sequences, and can be used to detect the 139A/G SNP of CYP2C8 gene. This study lays experimental foundation for the rapid and high- throughput SNP detection.

关 键 词：分子信标 断接式发夹探针 单核苷酸多态性 CYP2C8基因 

分 类 号：R394-33[医药卫生—医学遗传学] R446[医药卫生—基础医学]