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[目的]优化山楂PCR-SSCP反应体系与反应条件。[方法]以山楂为研究材料,采用改良的CTAB法分步提取叶绿体DNA和核DNA。从8对候选引物中分别筛选适合对叶绿体DNA和核DNA进行PCR扩增的引物,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]筛选出ropB、ropL、rpoC1、ITS2和psbA-trnH五对引物适用于山楂的PCR-SSCP分析。电泳时,上样缓冲液(不含甘油)与PCR产物等量混合,98℃碱(1%NaOH)变性15min,采用6%的聚丙烯酰胺凝胶(交联度29∶1),电泳缓冲液0.5×TBE,在4℃恒温和200 V恒压的条件下,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PCRSSCP最优反应体系与反应条件,为山楂SSCP分析奠定基础。
[Objective] The research aimed to optimize the PCR-SSCP reaction system and reaction conditions of hawthorn. [Method] With hawthorn as the research material, chloroplast DNA and nuclear DNA were extracted step by step by modified CTAB method. The primers suitable for PCR amplification of chloroplast DNA and nuclear DNA were screened from 8 candidate primers, and the PCR-SSCP reaction system and reaction conditions were optimized. PCR amplification products were detected by agarose gel electrophoresis, and denatured polyacrylamide gel electrophoresis was used to detect denatured PCR amplification products. [Result] Five pairs of primers ropB, ropL, rpoC1, ITS2 and psbA-trnH were selected for PCR-SSCP analysis of hawthorn. When electrophoresis, the sample buffer (without glycerol) was mixed with the PCR product in the same amount, denatured with 98% alkali (1% NaOH) for 15 minutes, and then separated by 6% polyacrylamide gel Liquid 0.5 × TBE, at 4 ℃ constant temperature and 200 V constant pressure conditions, electrophoresis 3 ~ 4 h, better hawthorn PCR-SSCP electrophoresis patterns can be obtained. [Conclusion] The optimal PCR reaction system and reaction conditions of hawthorn were established, which laid the foundation for SSCP analysis of hawthorn.