扩增条件对茶类植物RAPD带的影响

IMPACTS OF RAPD AMPLIFICATION CONDITIONS ON RAPD BANDS FOR CAMELLIA

采用梯度分析的方法试验了模板ＤＮＡ、引物、镁离子、ｄＮＴＰ和Ｔａｑ酶的浓度对茶类植物进行ＲＡＰＤ分析中ＤＮＡ扩增结果的影响。实验表明这些条件的变化对扩增出来的ＲＡＰＤ带的数目和强弱会产生影响。经过比较分析，筛选出对于茶类植物进行ＲＡＰＤ分析较理想的扩增条件：２．０ｍｍｏｌ／ＬＭｇＣｌ２，２００ｕｍｏｌ／ＬｄＮＴＰ，１５ｎｇ引物／２０ｕｌ反应体积，４ｎｇ模板ＤＮＡ／ｕｌ反应体积，１ＵＴａｑ酶／２０ｕｌ反应体积。

The impacts of the concentrations of template DNA, primer, magnesium, dNTPand Taq DNA polymerase on RAPD amplification for Camellia were tested by means ofladder analysis. The results showed that RAPD band patterns were changed observablywith the variation of concentrations of template DNA or primer or Taq DNA polymerase,and there was a positive interrelation between the both within a limited range, but littleimpact was generated by the concentrations of 1.0 - 4.0 mmol/L McCl2 or 100 - 400 umol/LdNTP on RAPD band patterns. The optimal amplification conditions for Camellia were chosenas follows: 4ng template DNA/ul reaCtion volume, 15 ng primer/20 ul reaction volume,2.0 mmol/L MgCl2, 200 Umol/L dNTP, 1 U Taq DNA polymerse/20 ul reaction volume.