非洲菊SRAP标记的DNA模板制备及反应体系优化

Template DNA and reaction system based on SRAP-PCR of Gerbera jamesonii

为获得非洲菊清晰的SRAP标记图谱,对非洲菊DNA的提取方法及其SRAP-PCR反应体系的影响因子进行了初步探讨,建立了扩增多态性高、重复性好、带型清晰的DNA模板和SRAP-PCR反应体系.最佳SPAR-PCR反应体系:在50μL的反应总体系中,Mg2+2.0mmol/L,dNTPs0.3mmol/L,DNA模板100ng,DNA聚合酶2.0U,上游、下游引物各0.22mmol/L.扩增程序:在94℃预变性4min,反应前5个循环在94℃变性1min,35℃复性1min,72℃延伸1min的条件下运行,随后的30个循环复性温度提高到55℃,最后72℃延伸5min.

To obtain clear sequence-related amplified polymorphism （SRAP） fingerprints of Gerbera jamesonii. ,method of DNA extraction and factors influencing SRAP analysis were studied. A reliable, effective and reproductive PCR reaction system for detecting SRAP was developed. Each 50 μL PCR reaction mixture consisted of 2.0 mmol/μL of Mg^2＋, 0.3 mmol/L of dNTPs, 100 ng of genomic DNA, 2.0 unit of Taq polymerase and 0.22 mmol/L of primer. Samples were subjected to thermal profile for amplification in an oven thermocycler： 4 min of denaturing at 94 ℃, five cycles of three steps, 1 min of denaturing at 94 ℃, 1 min of annealing at 35 ℃ and 1 min of elongation at 72 ℃, in the following 30 cycles the annealing temperature was increased to 55 ℃, with a final elongation step of 5 min at 72 ℃.