杨楠楠,李贵芳,徐琴,罗振元,李頔,熊永红,王仕敏,黄盛文
YANG Nannan,LI Guifang,XU Qin,LUO Zhenyuan,LI Di,XIONG Yonghong,WANG Shimin,HUANG Shengwen

• 1:贵州医科大学医学检验学院
• 2:贵州医科大学
• 3:贵州省人民医院检验科
• 4:遵义医学院检验系

摘要(Abstract)：

目的:采用焦磷酸测序定量检测人体γ珠蛋白基因(HBG)启动子区甲基化。方法:收集30例经基因检测确诊为轻型β-地中海贫血(简称地贫)患者的全血DNA样本,查找γ珠蛋白基因的启动子区Cp G位点,设计PCR扩增引物及焦磷酸测序引物;DNA样本经亚硫酸氢盐转化后进行PCR扩增,对PCR产物作单链处理,在测序引物的引导下进行焦磷酸测序,对Cp G位点的甲基化程度进行定量分析。结果:30例轻型β-地贫患者的HBG启动子区共150个Cp G位点均得到了甲基化程度的定量结果,其中-53、-50、+5、+16及+49位点高甲基化分别有28例、24例、13例、0例及7例,中甲基化分别有2例、6例、17例、30例及23例,未检测到低甲基化。结论:焦磷酸测序可对HBG启动子Cp G位点的甲基化程度进行定量检测。
Objective: To establish quantitative detection of methylation of γ-globin gene( HBG)promoter by pyrosequencing. Methods: The whole blood DNA samples from 30 patients with mild β-thalassemia diagnosed by gene detection were collected to find the Cp G site in the promoter region of γ-globin gene. PCR amplification primers and pyrosequencing primers were designed. DNA samples were amplified by PCR after transformation by hydrogen sulfite. The PCR products were treated with single strand,followed by pyrosequencing under the guidance of sequencing primers. The methylation degree of Cp G sites was quantitatively analyzed. Results: A total of 150 Cp G loci in HBG promoter region of 30 cases of mild thalassemia patients got quantitative results of methylation level. Among them,there were 28,24,13,0,7 cases of hypermethylation at-53,-50, + 5、+ 16 and + 49 respectively; There were 2,6,17,30,23 cases of middle methylation at them respectively; No hypomethylation was detected for all of them. Conclusion: Pyrosequencing can be used to quantify the degree of methylation at the Cp G site of HBG promoter.

关键词(KeyWords)： γ珠蛋白;基因;启动子区;甲基化;亚硫酸氢盐转化;焦磷酸测序
γ-globin;gene;promoter region;methylation;bisulphite transformation;pyrosequencing

Abstract:

Keywords:

基金项目(Foundation): 贵州省科技计划项目[黔科合平台人才(2016)5670号];; 贵州省科技合作计划项目[黔科合LH字(2015)7134号];; 贵州省留学人员科技创新项目[黔人项目资助合同(2016)15号];; 贵阳市科技创新平台计划[(20161001)35号]

作者(Author): 杨楠楠,李贵芳,徐琴,罗振元,李頔,熊永红,王仕敏,黄盛文
YANG Nannan,LI Guifang,XU Qin,LUO Zhenyuan,LI Di,XIONG Yonghong,WANG Shimin,HUANG Shengwen

DOI: 10.19367/j.cnki.1000-2707.2018.03.006

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