【摘要】 目的探讨聚合酶链反应-限制性片段长度多态性方法(PCR-RFLP)检测PARL基因Leu262Val多态性的最适实验条件.方法在PCR实验中,运用降落PCR(touchdown PCR,TD-PCR)方法优化PCR条件;对引物终浓度设定0.8μmol/L、0.6μmol/L、0.4μmol/L和0.32μmol/L四个浓度梯度进行扩增;通过2%琼脂糖凝胶电泳观察结果.在RFLP实验中,在内切酶量10 U不变的情况下,PCR产物量分别设定为5μL、10μL、15μL;酶切时间分别设定为12 h、8 h、4 h、2 h、1 h酶切,反应完毕后通过2%琼脂糖凝胶电泳观察结果.结果 (1)降落PCR方法能有效降低或避免非特异性扩增;(2)引物浓度为0.4μmol/L时,PCR产物量高且引物二聚体较少;(3)10μL的PCR产物进行酶切,电泳结果清晰;(4)在37℃条件下酶切4 h能完全消化一个酶切体系中的PCR产物.结论 PCR实验中,TD-PCR优化了PCR条件,为扩增目的条带提供了快速、特异、可靠的方法,最适引物浓度为0.4μm/L;RFLP实验中,最有效酶切方案为20μL的体系中加10μL产物和10 U内切酶,37℃消化4 h.更多还原

【Abstract】 Objective To determine the optimum experimental conditions of the Leu262Val polymorphism of PARL gene detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Method In the PCR,PCR program was optimized by touchdown PCR(TD-PCR);four kinds of primers’ concentration(0.32 umol/l,0.4 μmol/L,0.6 μmol/L,0.8 μmol/L)were used,PCR products were observed in 2% agarose.In the RFLP,three kinds of PCR product quantity(5 μL,10 μL,15 μL) and five kinds of incubation time(12 h,8 h,4 h,2 h,1 h) were set when the incision enzyme quantity was unchanging,PCR products were observed in 2% agarose.Results(1)TD-PCR showed no non-specific band and simplified the process for seeking the optimum annealing temperature in ordinary amplification.(2)When the primers concentration was 0.4μmol/L,PCR production level was higher than others and had less dimer of primers.(3)It was 10μl PCR production amount that was digested could creat a clear digested electropherogram.(4) It was 4 hours that was demanded in incubated reaction at 37 ℃.Conclusions The design of primers is the key of PCR,the optimum primer’s concentration is 0.4 μmol/L.TD-PCR can optimize connditions of ordinary PCR,it is more simple and effective than the ordinary PCR;The effective RFLP protocol:the system is 20 μL including 10 μL PCR reaction mixture and 10 U enzyme are incubated at 37 ℃ for 4 hours.更多还原