兰属SRAP-PCR反应体系的建立与优化

Establishment and Optimization of SRAP-PCR Reaction System in Cymbidium

为获得兰属清晰的SRAP标记图谱,对兰属SRAP-PCR反应体系进行了初步探讨,建立了扩增多态性高、重复性好、带型清晰的SRAP-PCR反应体系。最佳反应体系:在30μL反应总体系中,Mg2+2.2 mmol/L、dNTPs 0.8 mmol/L、DNA模板150 ng、DNA聚合酶2.0 U,上、下游引物各1.5μmol/L;扩增程序:在94℃预变性4 min,反应前5个循环在94℃变性1 min、35℃复性1 min、72℃延伸1 min的条件下运行,随后的30个循环复性温度提高至55℃,最后72℃延伸5 min.

To obtain clear Sequence-related amplified polymorphism （SRAP） fingerprints of Cymbidium, the factors influencing SRAP analysis were studied. A reliable, effective and reproductive PCR reaction system for detecting SRAP was developed. Each 30 μL PCR reaction mixture consisted of 2.2 mmol/pL of Mg2＋, 0.8 mmol/L of dNTPs, 150 ng of genomic DNA, 1.5 pmol/L of primer and 2.0 unit of Taq polymerase. Samples were subjected to thermal profile for amplification in an oven thermocycler： 4 min of denaturing at 94 ℃, five cycles of three steps, 1 rain 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 rain at 72 ℃.