应用RAPD技术构建绞股蓝DNA指纹图谱

Construction of DNA fingerprint for Gynostemma Pentaphyllum (Thunb) Makino based on RAPD analysis

以不同类型绞股蓝(Gynostemma Pentaphyllum,(Thunb)Makino)DNA为模板,进行RAPD反应条件的优化及RAPD结果分析.最终筛选的RAPD反应条件为:20μL PCR反应体系中包括了10×buffer2.0μL,Mg2+2.0 mmol/L,dNTP 0.2 mmol/L,引物0.5 mmol/L,Taq酶1U,模板DNA 50 ng.反应程序为94℃3 min→(94℃30 s→38℃40 s→72℃1 min)45个循环→72℃10min→4℃保持,并从66条随机引物中筛选出5条带型丰富、适合于绞股蓝分析的随机引物.以这5条随机引物对不同类型绞股蓝进行RAPD扩增,识别其特征性多态位点,建立分子标记检索表.

The optimized RAPD analysis with different Gynostemma Pentaphyllum as templates in PCR amplitication was reported in this paper. The optional parameters for 20 μL PCR reaction system are 10 × buffer 2.0 μL, Mg^2＋ 2 mmol/L, dNTP 0.2 mmol/L, random primer 0.5 mmol/L, Taq DNA polymerase 1U, and template DNA 50ng, respectively. The optimized protocol for the reaction is 94℃ （ 3 min） → 94℃ （ 30s ） → 38℃ （ 40s ）→ 72℃ （ 1 min ） 45 cycles→ 72℃ （ 10 min ） → 4℃ （ constant temperature）. The 5 primers were selected from 66 random ones to carry out this RAPD amplification so that the characteristic polymorphous sites of Gynostemma Pentaphyllum are located as well as the identification key based on molecular markers is established.