山楂PCR－SSCP反应体系与反应条件的优化

[目的]优化山楂PCR-SSCP反应体系与反应条件。[方法]以山楂为材料,采用改良的CTAB法分步提取叶绿体DNA和核DNA,对PCR-SSCP引物进行筛选,同时进行PCR-SSCP反应体系和反应条件的优化,琼脂糖凝胶电泳检测PCR扩增产物,非变性聚丙烯酰胺凝胶电泳检测变性后的PCR扩增产物。[结果]rop B、rop L、rpo C1、ITS2、psb A-trn H 5对引物适用于山楂PCR-SSCP分析。电泳时,采用6%聚丙烯酰胺凝胶(交联度29∶1),4℃恒温,200 V恒压,上样缓冲液(不含甘油)与PCR产物等量混合,98℃碱(1%Na OH)变性15 min,电泳缓冲液为0.5×TBE,电泳3~4 h可得到较好的山楂PCR-SSCP电泳图谱。[结论]建立了山楂PCR-SSCP最优反应体系与反应条件,为山楂SSCP分析奠定基础。

Optimization of PCR-SSCP Reaction System and Reaction Condition in Crataegus spp.

Objective] The aim was to optimize PCR-SSCP reaction system and reaction condition.[ Method] The chloroplast DNA and nu-clear DNA in Crataegus spp.were step-by-step extracted by improved CTAB method, the PCR-SSCP primers were selected, and the PCR-SS-CP reaction system and reaction conditions were optimizated.The PCR-SSCP productions were tested by agarose gelelectrophoresis, the dena-tured PCR-SSCP products were analyzed by native polyacrylamide gel.[ Result] The results showed that five pairs primers ( psbA-trnH、ropB、ropL、rpoC1、ITS2) of all are suitale for PCR-SSCP in Crataegus spp..The clear electrophoretogram of PCR-SSCP in Crataegus spp.were ob-tained through using 6% native polyacrylamide gel.(Crosslinking degree is 29∶1) at 4 ℃and 200 V for 3-4 h, with the volume of the load-ing buffer ( without glycerol) was same with the volume of PCR products, denaturation temperature was 98 ℃ under 1%NaOH, denaturetion time was 15 minutes, electrophoresis buffer was 0.5 ×TBE.[ Conclusion] The study optimize PCR-SSCP reaction system and reaction condi-tion, in order to lay the foundation for optimization of hawthorn SSCP analysis.