摘要
目的建立食管癌易感基因乙醛脱氢酶2(ALDH2)编码区SNP rs671的LATE(Linear-After-The-Exponential)-PCR非标探针分型技术。方法用Chelex法提取口腔拭子DNA,LATE-PCR技术制备单链,等位基因特异性探针与单链杂交后,在荧光定量PCR仪上通过链融解曲线分析,根据融链峰进行SNP分型。用经PCR-RFLP法分型的已知样本验证本方法的有效性。结果LATE-PCR产物直接链融解曲线分析可以区分GG型和A基因携带者。PCR后加入非封闭探针分析可以鉴别3种基因型。15 bp 3’末端C6氨基修饰的封闭探针,可以在PCR前加入体系,A型、G型任一探针即可准确地鉴别3种基因型。结论LATE-PCR结合非标探针技术,可以实现SNP rs671的简便、快速、闭管分型,可用于食管癌高危个体筛查、法医嫌疑人排查等。
Objective To develop a novel method,which is LATE(Linear-After-The-Exponential)-PCR and unlabelled DNA Probes based,for genotyping SNP rs671 in aldehyde dehydrogenase-2(ALDH2)gene.Methods With tempelate DNA prepared by Chelex method,the single strand DNA were generated by LATE-PCR.Then the DNA probes annealed,the hybrids were melted on a real-time PCR cycler.The melting peaks and SNP genotypes were analyzed.Known samples genotyped by PCR-RFLP were used to verify the accuracy and reliability.Results Direct melting the PCR products can determine the GG type.When the non-modified probes were used,all 3 SNP genotypes can be determined.The modified probes can be added to the reaction tube before PCR and determine 3 SNP genotypes.Conclusion LATE-PCR coupled with unlabelled DNA probes,can genotype SNP rs671 in a closed tube manner,which is simple and fast,representing a novel method for searching the high-risk person of esophageal cancer or suspects in Forensic Science.
作者
杨斌
杨瑞
周路敏
张振辉
冯巍
赵贵森
YANG Bin;YANG Rui;ZHOU Lu-min;ZHANG Zhen-hui;FENG Wei;ZHAO Gui-sen(School of Forensic Medicine,Henan University of Science and Technology,Luoyang 471003,China)
出处
《食管疾病》
2020年第4期265-269,共5页
Journal of Esophageal Diseases
基金
河南省教育厅自然科学研究计划项目(2009B340001)
河南科技大学实验技术开发基金项目(SY1920021)。
作者简介
杨斌(1989-),男,河南辉县人,从事法医遗传学分析研究;通信作者:赵贵森,男,博士,副教授,E-mail:lyfy@haust.edu.cn。
