PCR-RFLP法检测DDAH2基因rs805304多态性的实验条件研究
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摘要
目的探讨聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测二甲基精氨酸二甲胺水解酶2(DDAH2)rs805304多态性的最适实验条件。方法运用PCR-RFLP技术分别对2016年1月～2017年3月昆明医科大学第一附属医院收治的182例2型糖尿病患者和147名健康体检者DDAH2基因rs805304的多态性进行检测,对影响PCR-RFLP方法的主要因素进行研究。结果最适实验条件:退火温度为58℃,变性温度为95℃,2×Power Taq PCR Master Mix浓度为12.5μL,引物浓度为0.8μmo/L,酶切体系为15μL体系中加8.0μL产物,用5 U的限制性内切酶Bst UI消化。结论最适实验条件的摸索为后续研究奠定了基础。
Objective To investigate the optimum experimental conditions for dimethylarginine dimethylamine hydrolase 2(DDAH2) gene rs805304 polymorphism by polymerase chain reaction-restricted fragment length polymorphism(PCR-RFLP). Methods The DDAH2-rs805304 locus polymorphism in 182 T2 DM cases admitted to the First Affiliated Hospital of Kunming Medical University from January 2016 to March 2017 and 147 healthy controls were detected by PCR-RFLP. The main factors influencing PCR-RFLP were studied. Results The optimum conditions: annealing temperature was 58℃, denaturation temperature was 95℃, 2 × Power Taq PCR Master Mix concentration was 12.5 μL,primer concentration was 0.8 μmo/L, the effective system was 15 μL including 8 μL DNA solution and 5 U restriction enzyme BstUI. Conclusion The exploration of optimal experimental conditions has laid a foundation for the subsequent research.
Objective To investigate the optimum experimental conditions for dimethylarginine dimethylamine hydrolase 2(DDAH2) gene rs805304 polymorphism by polymerase chain reaction-restricted fragment length polymorphism(PCR-RFLP). Methods The DDAH2-rs805304 locus polymorphism in 182 T2 DM cases admitted to the First Affiliated Hospital of Kunming Medical University from January 2016 to March 2017 and 147 healthy controls were detected by PCR-RFLP. The main factors influencing PCR-RFLP were studied. Results The optimum conditions: annealing temperature was 58℃, denaturation temperature was 95℃, 2 × Power Taq PCR Master Mix concentration was 12.5 μL,primer concentration was 0.8 μmo/L, the effective system was 15 μL including 8 μL DNA solution and 5 U restriction enzyme BstUI. Conclusion The exploration of optimal experimental conditions has laid a foundation for the subsequent research.
引文
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[20]黄立华,王亚琴.分子生物学实验技术.基础与拓展[M].北京:科学出版社,2018:47.
[2]Lu TM,Lin SJ,Lin MW,et al.The association of dimethylarginine dimethylaminohydrolase1 gene polymorphism with type 2 diabetes:a cohort study[J].Cardiovasc Diabetol,2011,10:16.
[3]Lai L,Ghebremariam YT.Modulating DDAH/NOS Pathway to Discover Vasoprotective Insulin Sensitizers[J].JDiabetes Res,2016,2016:1982096.
[4]Zheng J,Wang K,Jin P,et al.The association of adiposederived dimethylarginine dimethylaminohydrolase-2 with insulin sensitivity in experimental type 2 diabetes mellitus[J]Acta Biochim Biophys Sin(Shanghai),2013,45(8):641-648.
[5]Yuan Q,Hu CP,Gong ZC,et al.Accelerated onset of senescence of endothelial progenitor cells in patients with type2 diabetes mellitus:Role of dimethylarginine dimethylaminohydrolase 2 and asymmetric dimethylarginine[J].Biochem Biophys Res Commun,2015,458(4):869-876.
[6]伊桐凝,张锦,于世家.阿司匹林抗高糖诱导的内皮细胞衰老过程中对DDAH-ADMA系统及Caveolin-1蛋白的影响[J].中国动脉硬化杂志,2016,26(5):469-473.
[7]Liu X,Hou L,Xu D,et al.Effect of asymmetric dimethylarginine(ADMA)on heart failure development[J].Nitric Oxide,2016,54:73-81.
[8]Marra M,Marchegiani F,Ceriello A,et al.Chronic renal impairment and DDAH2-1151 A/C polymorphism determine ADMA levels in type 2 diabetic subjects[J].Nephrol Dial Transplant,2013,28(4):964-971.
[9]Fogarty RD,Abhary S,Javadiyan S,et al.Relationship between ddah gene variants and serum adma level in individuals with type 1 diabetes[J].J Diabetes Complications,2012,26(3):195-198.
[10]Tanhauserova V,Tomandl J,Pacal L,et al.ADMA,SDMAand L-arginine/ADMA ratio but not DDAH genetic polymorphisms are reliable predictors of diabetic nephropathy progression as identified by competing risk analysis[J].Kidney Blood Press Res,2012,36(1):200-208.
[11]Maasa R,Erdmannb J,Lüneburgc N,et al.Polymorphisms in the promoter region of the dimethylarginine dimethylaminohydrolase 2 gene are associated with prevalence of hypertension[J].Pharmacol Res,2009,60(6):488-493.
[12]Bai Y,Chen J,Sun K,et al.Common genetic variation in DDAH2 Is associated with intracerebral hemorrhage in Chinese population:a Multi-centercase-control study in China[J].Clin Sci,2009,117(7):273-279.
[13]Kim HY,Kim HS.Dimethylarginine dimethylaminohydrolase mediates inhibitory effect of interleukin-10 on angiotensinⅡ-induced hypertensive effects in vascular smooth muscle cells of spontaneously hypertensive rats[J].Cytokine,2016,77:203-210.
[14]Andreozzi F,Presta I,Mannino GC,et al.A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with insulin sensitivity[J].PLo SOne,2012,7(4):e36224.
[15]高胜利,翟晓娟,李莉,等.围产期高盐饮食对雄性子代大鼠肠系膜动脉DDAH2/ADMA/e NOS/NO通路的影响[J].中国动脉硬化杂志,2016,24(5):463-468.
[16]李芸.聚合酶链反应在医学检验中的应用和进展[J].中国城乡企业卫生,2017,8(8):18-20.
[17]史慧明.聚合酶链反应(PCR)试验的方法及临床意义[J].世界最新医学信息文摘,2016,18(3):126-128.
[18]宋方洲,何凤田.生物化学与分子生物学实验[M].北京:科学出版社,2008:135-138.
[19]刘长霞,罗施中.分子生物学实验技术[M].北京:化学工业出版社,2018:69.
[20]黄立华,王亚琴.分子生物学实验技术.基础与拓展[M].北京:科学出版社,2018:47.