CAJ | 학술논문

以黄鳝基因组DNA为模板,采用正交试验设计方法,对各反应因子、引物退火温度和循环参数进行优化.建立了黄鳝的最适ISSR-PCR反应体系,25 μl反应体系中含2.5 mmol/ L Mg2+,250 μmol/ L dNTPs,0.25 μmol/ L 引物,1.0 U Taq DNA聚合酶,30 ng DNA模板.最佳反应程序:94℃预变性5 min;94℃变性40 s,48～57℃复性40 s(随引物而确定),72℃延伸1.5 min,循环次数40;72℃延伸10 min.利用所建立的ISSR反应体系,获得了清晰、重复性好、多态性高的DNA谱带.
이황선기인조DNA위모판,채용정교시험설계방법,대각반응인자、인물퇴화온도화순배삼수진행우화.건립료황선적최괄ISSR-PCR반응체계,25 μl반응체계중함2.5 mmol/ L Mg2+,250 μmol/ L dNTPs,0.25 μmol/ L 인물,1.0 U Taq DNA취합매,30 ng DNA모판.최가반응정서:94℃예변성5 min;94℃변성40 s,48～57℃복성40 s(수인물이학정),72℃연신1.5 min,순배차수40;72℃연신10 min.이용소건립적ISSR반응체계,획득료청석、중복성호、다태성고적DNA보대.
Based on the genomic DNA of Monopterus albus, the factors influencing ISSR-PCR system were explored with the orthogonal design. The optimized ISSR-PCR system (25 μl) was determined as follows: 2.5 mmol / L Mg2+, 250 μmol / L dNTPs, 0.25 μmol / L primers, 1.0 U Taq DNA polymerase, 30 ng DNA template. The reaction program was set including initial denaturation for 5 minutes at 94℃, 40 cycles of denaturation for 40 seconds at 94℃,annealing for 40 seconds at 47 ～57℃, extension for 1.5 minutes at 72℃,with a final extension of 10 minutes at 72℃. The clear and reproducible amplification was obtained by the established ISSR reaction system.