松毛虫mtDNA PCR反应体系优化

为了应用线粒体DNA研究松毛虫种群的遗传结构,本研究采用正交试验设计法L16(45)对影响松毛虫mtDNA PCR反应体系的5个因素(Taq酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化试验并对PCR扩增程序中的退火温度及循环次数进行了单因素梯度比较优化。建立了松毛虫mtDNA PCR反应的最佳体系即在50μL体系中含模板150 ng、0.10μmol/L引物、0.4 mmol/L dNTP、1.0UTaq DNA聚合酶、2.5 mmol/LMg2+。同时通过PCR比较,确定最佳循环次数为35个循环,最佳退火温度为56℃,获得了松毛虫mtDNA PCR反应的最佳扩增程序。

Optimization of PCR reaction system of Pine caterpillars mtDNA

To examine the genetic structure of Pine caterpillars based on mtDNA,the orthogonal design was used to optimize mtDNA PCR reaction system for P.caterpillars using five factors(Taq DNA polymerase,Mg2+,DNA template,dNTP and primer) at four levels,respectively.Annealing temperature and cycle times affecting the mtDNA PCR researching system were optimized by single factor experiment.And a most suitable PCR system for P.caterpillars was established.The 50 μL volume system contained 150 ng DNA template,0.10 μmol/L primers,0.4 mmol/L dNTP,1.0 U Taq DNA polymerase and 2.5 mmol/L Mg2+.The better number of cycles were 35.Better annealing temperatures is 56 ℃ for PCR reaction.The result provided a standardizing mtDNA PCR program for P.caterpillars.