从血液和唾液中自动化提取人体DNA的探索与应用

Exploration and application of automatic extraction of human DNA from blood and saliva

目的从样本获取方式、储存条件和DNA提取方法等方面探索快速、有效、低成本、无创伤、适合于大规模流行病学研究的基因组DNA来源样本。方法对带有分离胶的血细胞、EDTA抗凝全血、分离血清后的凝固血细胞、新鲜唾液、-20℃保存1个月的唾液、2种唾液保护液作用的唾液样本和棉拭子获得的口腔黏膜上皮细胞样本进行DNA提取和检测,验证不同样本获得DNA的稳定性。随机抽取1人的2份质量控制合格的DNA样品,采用高通量测序技术对亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性位点进行分型检测。结果带有分离胶的血细胞样本中,分离胶对血细胞DNA的提取效果影响明显,DNA样品合格率为37.7%(1130/3000)。EDTA抗凝全血样本DNA提取效果良好,DNA浓度为(180.20±20.30)mg/L,D260/D280为1.90±0.10。分离血清后的凝固血细胞样本中DNA降解严重,提取的DNA浓度为28.91~34.53 mg/L。新鲜唾液和-20℃保存1个月的唾液样本经提取后得到的DNA浓度均合格,且差异无统计学意义(P>0.05)。2种唾液保护液作用下的唾液样本提取得到的DNA浓度均合格,且差异无统计学意义(P>0.05)。口腔黏膜上皮细胞样本提取的DNA浓度为(48.41±9.81)mg/L,低于唾液样本来源的DNA浓度。高通量测序结果显示,按照质量控制标准筛选的合格DNA样品可以实现体外目的基因片段的扩增和MTHFR C677T基因多态性位点的分型检测。结论血液和唾液样本均是人体基因组DNA提取的良好来源,唾液样本成本低、采集无创伤且质量控制合格,是一种更有效的DNA来源样本。

Objective To search for a genomic DNA extraction sample source characterized by rapid,effective,low cost,noninvasive,and suitable for large-scale epidemiological studies considering sample acquisition,storage and DNA extraction methods.Methods DNA samples were extracted and detected from blood cells with separating gel,ethylenediaminetetraacetic acid(EDTA)anticoagulant blood,coagulated blood cells after serum separation,fresh saliva,saliva stored at-20℃for 1 month,saliva with 2 kinds of saliva protective solutions and oral mucosal epithelial cell samples obtained by cotton swab.The stability of genomic DNA obtained from different samples were verified.Two DNA samples with eligible quality control were randomly selected from one subject,and methylenetetrahydrofolate reductase(MTHFR)C677T gene polymorphism was genotyped using high-throughput sequencing.Results Separating gel seriously reduced the DNA extraction effect,and the qualified rate of DNA samples was 37.7%(1130/3000)in blood cells with separating gel.The DNA extraction efficiency from EDTA anticoagulant blood was good,with the DNA concentration being(180.20±20.30)mg/L and D260/D280 being 1.90±0.10.In coagulated blood cells after serum separation,remarkable DNA degradation was found,with the DNA concentration being 28.91-34.53 mg/L.The concentrations of DNA extracted from fresh saliva and saliva stored at-20℃for 1 month were both qualified without significant difference(P>0.05).The concentrations of DNA extracted from saliva with 2 kinds of protective solutions were also qualified without significant difference(P>0.05).The concentration of DNA extracted from oral mucosal epithelial cells was(48.41±9.81)mg/L,which was lower than that from saliva samples.High-throughput sequencing showed that amplification of targeted gene fragments in vitro and genotyping detection of polymorphic sites of the MTHFR C677T gene could be achieved using the qualified DNA samples screened according to the quality control criteria.Conclusion Blood and saliva are eligible samples for extracting genomic DNA.Saliva has served as a better source sample to obtain human genomic DNA with low cost,no trauma and good quality.