多倍体罗汉果PCR-RFLP参数的优化与应用

Optimization and preliminary study on the PCR-RFLP parameter of polyploidy Siraitia grosvenorii

关键词:  多倍体罗汉果  PCR-RFLP  酶切反应  优化与应用

Key words:  polyploidy Siraitia grosvenorii  PCR-RFLP  digestion reaction  optimization and application

摘要:

以多倍体罗汉果DNA为材料,采用L16(4⁵)正交组合试验和单因素梯度试验,研究Mg²⁺、dNTP、引物、Taq DNA聚合酶、模板DNA浓度和退火温度、循环次数等对PCR扩增结果以及内切酶量、酶切时间对酶切反应的影响。结果表明,多倍体罗汉果RFLP最优PCR反应体系和扩增参数为:在25 μL扩增反应体系中,10×Buffer 2.5 μL,MgCl₂ 1.5 mmol/L,dNTP 0.2 mmol/L,引物0.1 μmol/L,Taq DNA聚合酶2.0 U,模板DNA 60 ng; 退火温度为56 ℃,循环次数为35次。酶切反应体系:内切酶10×Buffer 2.0 μL,内切酶5.0 U,PCR产物15 μL,超纯水补至20 μL; 酶切时间2 h。

Abstract:

In this paper,the DNA of polyploidy Siraitia grosvenorii was regarded as the research object,several important factors including MgCl₂,dNTP,primer,Taq DNA polymerase,template DNA,annealing temperature,amplification and enzyme quantity,digestion reaction time were optimized by L16(4⁵)orthogonal design experiment and single factor experiment in order to investigate the effects on the results of PCR amplification and digestion reaction. The test results showed that a total volume of 25 μL PCR reaction system contained 10×Buffer 2.5 μL,MgCl₂ 1.5 mmol/L,dNTP 0.2 mmol/L,general service primer 0.1 μmol/L,Taq DNA polymerase 2.0 U,template DNA 60 ng,and annealing temperature was 56 ℃,amplification for 35 cycles was optimizational PCR amplification reaction of RFLP of polyploidy S.grosvenorii. The total volume of 20 μL digestion reaction system contained digestion enzyme 10×Buffer 2.0 μL,digestion enzyme 5.0 U,PCR product 15 μL,digestion reaction time 2 h.