苦楝SRAP-PCR反应体系的建立及优化

    Establishment and optimization of SRAP-PCR system in Melia azedarach

    • 摘要:
      目的 应用SRAP技术对苦楝Melia azedarach遗传多样性、种质资源鉴定等开展研究,对苦楝SRAP-PCR体系中的模板DNA、dNTPs、Mg2+、引物和Taq DNA聚合酶5个组分浓度进行优化,建立适合苦楝的SRAP-PCR反应体系.
      方法 采用单因素试验对反应体系中的5个因素分别设置8个浓度梯度水平,确定浓度范围后进行L16(45)正交试验设计,并对结果进行打分,确定优化体系.
      结果和结论 SRAP对苦楝DNA浓度的要求不高,有一个较宽的浓度适宜范围;dNTPs在0.1~0.2 mmol·L-1范围内,能扩增出条带基本相同的清晰谱带;Mg2+为2.0 mmol·L-1左右时扩增条带较清晰且数量多;引物介于0.48~0.64 μmol·L-1均能扩增出带型基本保持一致且清晰的谱带;Taq DNA聚合酶在0.50~2.00 U范围内可以得到清晰的带型.根据正交试验设计16个处理的得分,确定优化的反应体系为:模板DNA 30 ng、dNTPs 0.125 mmol·L-1、Mg2+ 2.25 mmol·L-1、引物0.48 μmol·L-1Taq DNA聚合酶0.75 U,反应总体积25 μL.

       

      Abstract:
      Objective In order to study the genetic diversity and evaluate the germplasm resources of Melia azedarach using SRAP markers, the concentration of DNA template, dNTPs, Mg2+, primer and Taq polymerase were tested to determine their optimal levels and establish the optimal SRAP reaction system in M. azedarach.
      Method Five factors, each with 8 concentration levels, were conducted to screen their feasible concentration range using single factor experiment, and then the orthogonal experiment L16(45) was carried out and the optimized system was determined.
      Result and conclusion The concentration range of DNA template was wider in SRAP system in M. azedarach. When the concentration of dNTPs ranged from 0.1 to 0.2 mmol·L-1, the bands were steady; when the concentration of Mg2+ was around 2.0 mmol·L-1, more bands were amplified and clear. The band type was consistent and clear when the primer concentration ranged from 0.48 to 0.64 μmol·L-1. When the Taq polymerase ranged from 0.50 to 2.00 U, more bands were amplified and clear. The optimum SRAP-PCR system was established on the basis of orthogonal experiment, which was DNA template 30 ng, dNTPs 0.125 mmol·L-1, Mg2+ 2.25 mmol·L-1, primers 0.48 μmol·L-1, and Taq DNA polymerase 0.75 U in the 25 μL reaction system.

       

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