草鱼TRAP-PCR反应体系的建立

Establishment of TRAP-PCR Reaction Conditions in Grass Carp

目的:通过优化草鱼TRAP-PCR反应体系,将新型分子标记-靶位区域扩增多态性(target region amplified polymorphism,TRAP)引用到草鱼遗传多样性研究中。方法:以草鱼DNA为材料,分析了模板DNA、Mg2+、dNTPs、引物浓度,以及循环参数、退火温度对TRAP-PCR扩增结果的影响。结果:确立了稳定性强、重复性好的草鱼TRAP-PCR最佳反应体系和扩增参数:在25μl的PCR反应体系中,含约50ng模板DNA,1UTaq酶,1×PCR缓冲液,2.0mmol/L MgCl2,4种dNTPs各0.2mmol/L,固定引物与随机引物各15pmol;首先使模板在94℃变性3min;然后94℃变性1min,38℃退火1min,72℃延伸lmin进行5个循环;接着94℃变性45s,55℃退火45s,72℃延伸lmin再进行35个循环,最后72℃延伸7min。结论:TRAP-PCR反应体系稳定可靠,该新型分子标记可应用于草鱼遗传多样性研究中。

Objectives： In order to apply a new kind of molecular marker, which is target region amplified polymorphiam （TRAP）, to grass carp genetic analysis. In this paper, the influencing factors of target region amplified polymorphism （TRAP） were analyzed. Methods： By using genomic DNA of grass carp （ Ctenopharyngodon idella）, adjusting the concentration of template DNA, Mg^2＋ , dNTPs, primers and the parameters of cycles to select the best optimization. Results： The stable and reproducible optimum reaction system of TRAP-PCR amplification parameters were established for grass carp. The optimal system was in 25μl reaction volume containing about 50 ng template DNA, 1U Taq polymerase, 1 × PCR Buffer, 2.0mmol/L MgCl2 ,0.2mmol/L dNTPs and 15pmol of each primer.The amplification program was after 1 cycle initial denaturation at 94℃ for 3 min,each 5 cycles consisted of 94℃ denaturation for 1 min,38℃ annealing for 1 min,72℃ extension for 1 min,then each 35 cycles consisted of 94℃ denaturation for 45s,55℃ annealing for 45s,72℃ extension for 1 min and finally extension was at 72 ℃for 7 min. Condusion： The - PCR amplification was stable and reproducible, this new kind of molecular was useful for grass carp genetic diversity analysis.