基于改进的DNA连接酶链式反应发现NOD2基因多态性与冠心病的相关性

发布时间：2018-02-25 09:27

本文关键词： 冠状动脉疾病基因分型 DNA连接酶类多态性单核苷酸　出处：《中国循环杂志》2017年06期 　论文类型：期刊论文

【摘要】：目的:本研究改进基于脱氧核糖核酸(DNA)连接酶的中低通量基因分型方法,并用此方法进行核苷酸结合寡聚结构域(NOD)样受体基因NOD1和NOD2与冠心病的关联分析。方法:通过多重聚合酶链式反应(PCR)预扩增增加连接模板分子数量,提高特异性连接效率,优化DNA探针设计,摸索连接反应温度、时间、循环圈数及连接酶种类实现等位基因特异性连接,通过荧光掺入PCR和毛细管电泳实现多种等位基因特异性产物一次性检测。Sanger测序验证此方法准确性后,利用此方法对NOD1和NOD2基因上的单核苷酸多态性(SNP)位点在1 555例冠心病患者和1887例对照受试者中进行分型和关联分析。结果:通过优化反应条件和等位基因特异性探针设计原则,实现10 ng DNA样本一次性分型30个等位基因多态性位点。基于改进的DNA连接酶中低通量基因分型方法,NOD1、NOD2基因与冠心病的关联分析发现,NOD2基因上rs1861759和rs751271位点在非高血压条件下与冠心病相关(P均0.05),经Bonferroni多重检验校正后,相关性仍然显著(P均0.05)。结论:本研究优化了DNA连接酶链式反应技术在设计上的关键点,联合使用多重PCR和毛细管电泳,建立了一种高准确性、低成本、满足基于中低通量位点分型的临床分子诊断和科研需求的基因分型新方法,并用该方法发现NOD2基因上的位点rs1861759和rs751271在非高血压条件下与冠心病相关。
[Abstract]:Objective: to improve the method of low and low throughput genotyping based on DNA ligase. The association of nucleotide binding oligodeoxyribonucleotide oligodeoxyribonucleotide receptor genes (NOD1 and NOD2) with coronary heart disease (CHD) was analyzed. Methods: the number of ligated template molecules was increased by multiplex polymerase chain reaction (PCR) and the specific binding efficiency was improved. To optimize the design of DNA probe, explore the reaction temperature, time, cycle number and the type of ligase to realize the allele-specific connection. Multiple allele-specific products were detected by fluorescence incorporation of PCR and capillary electrophoresis, and Sanger sequencing was used to verify the accuracy of the method. The single nucleotide polymorphisms (SNPs) of NOD1 and NOD2 genes were classified and analyzed in 1 555 patients with coronary heart disease and 1 887 control subjects by this method. Results: by optimizing the reaction conditions and the principle of allele specific probe design, There were 30 allelic polymorphism loci in 10 ng DNA samples. Based on the improved low throughput genotyping method of DNA ligase, the association analysis between NOD1 and NOD2 gene and coronary heart disease showed that the rs1861759 and rs751271 loci on NOD2 gene were in non-hyperglycemic blood. Under the pressure condition, the correlation P between coronary heart disease and coronary heart disease was 0.05g, which was corrected by Bonferroni multiple test. Conclusion: this study optimized the key point of DNA ligase chain reaction technology in design, combined with multiple PCR and capillary electrophoresis to establish a high accuracy and low cost. A new genotyping method for clinical molecular diagnosis and scientific research based on low and low throughput locus typing was developed. The rs1861759 and rs751271 loci on NOD2 gene were found to be associated with coronary heart disease under non-hypertensive conditions.
【作者单位】：首都医科大学附属北京安贞医院北京市心肺血管疾病研究所血管生物研究室;北京大学分子医学研究所;南昌大学人类衰老研究所生命科学学院;
【基金】：973项目(2013CB530700) 国家自然科学基金重点项目(81130003);国家自然科学基金面上项目(81070262)
【分类号】：R541.4

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