本文關(guān)鍵詞： 冠狀動脈疾病 基因分型 DNA連接酶類 多態(tài)性 單核苷酸　出處：《中國循環(huán)雜志》2017年06期 　論文類型：期刊論文

【摘要】：目的:本研究改進基于脫氧核糖核酸(DNA)連接酶的中低通量基因分型方法,并用此方法進行核苷酸結(jié)合寡聚結(jié)構(gòu)域(NOD)樣受體基因NOD1和NOD2與冠心病的關(guān)聯(lián)分析。方法:通過多重聚合酶鏈式反應(yīng)(PCR)預(yù)擴增增加連接模板分子數(shù)量,提高特異性連接效率,優(yōu)化DNA探針設(shè)計,摸索連接反應(yīng)溫度、時間、循環(huán)圈數(shù)及連接酶種類實現(xiàn)等位基因特異性連接,通過熒光摻入PCR和毛細管電泳實現(xiàn)多種等位基因特異性產(chǎn)物一次性檢測。Sanger測序驗證此方法準確性后,利用此方法對NOD1和NOD2基因上的單核苷酸多態(tài)性(SNP)位點在1 555例冠心病患者和1887例對照受試者中進行分型和關(guān)聯(lián)分析。結(jié)果:通過優(yōu)化反應(yīng)條件和等位基因特異性探針設(shè)計原則,實現(xiàn)10 ng DNA樣本一次性分型30個等位基因多態(tài)性位點�；诟倪M的DNA連接酶中低通量基因分型方法,NOD1、NOD2基因與冠心病的關(guān)聯(lián)分析發(fā)現(xiàn),NOD2基因上rs1861759和rs751271位點在非高血壓條件下與冠心病相關(guān)(P均0.05),經(jīng)Bonferroni多重檢驗校正后,相關(guān)性仍然顯著(P均0.05)。結(jié)論:本研究優(yōu)化了DNA連接酶鏈式反應(yīng)技術(shù)在設(shè)計上的關(guān)鍵點,聯(lián)合使用多重PCR和毛細管電泳,建立了一種高準確性、低成本、滿足基于中低通量位點分型的臨床分子診斷和科研需求的基因分型新方法,并用該方法發(fā)現(xiàn)NOD2基因上的位點rs1861759和rs751271在非高血壓條件下與冠心病相關(guān)。
[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.
【作者單位】： 首都醫(yī)科大學附屬北京安貞醫(yī)院北京市心肺血管疾病研究所血管生物研究室;北京大學分子醫(yī)學研究所;南昌大學人類衰老研究所生命科學學院;
【基金】：973項目(2013CB530700) 國家自然科學基金重點項目(81130003);國家自然科學基金面上項目(81070262)
【分類號】：R541.4

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