CAJ | 학술논문

采用正交设计法,从dNTPs浓度、引物浓度、Mg2＋浓度、Taq DNA聚合酶用量4个因素3个水平出发,优化设计圆尾鲎DNA的PCR反应体系（引物为中国鲎微卫星引物）.并采用直观分析方法分析正交试验结果,最终建立了圆尾鲎SSR-PCR最佳反应体系：总体积20μL,Taq DNA聚合酶1.5 U、dNTPs 0.16 mmol/L、引物0.2μmol/L、Mg2＋2.0 mmol/L;并通过PCR梯度实验进一步优化模板DNA质量浓度、退火温度及退火时间,获得最佳反应条件：模板DNA质量浓度为30 ng/μL,退火温度为48℃,退火时间为20～25 s.对最佳反应体系和反应条件进行了检验,结果显示该反应体系稳定性高、重复性好.
채용정교설계법,종dNTPs농도、인물농도、Mg2＋농도、Taq DNA취합매용량4개인소3개수평출발,우화설계원미후DNA적PCR반응체계（인물위중국후미위성인물）.병채용직관분석방법분석정교시험결과,최종건립료원미후SSR-PCR최가반응체계：총체적20μL,Taq DNA취합매1.5 U、dNTPs 0.16 mmol/L、인물0.2μmol/L、Mg2＋2.0 mmol/L;병통과PCR제도실험진일보우화모판DNA질량농도、퇴화온도급퇴화시간,획득최가반응조건：모판DNA질량농도위30 ng/μL,퇴화온도위48℃,퇴화시간위20～25 s.대최가반응체계화반응조건진행료검험,결과현시해반응체계은정성고、중복성호.
SSR-PCR reaction system in Carcinoscorpius rotundicauda using microsatellite primers from Tachypleus tridentatus was constructed and optimized by employing L9（34）orthogonal design with three levels and four factors,which were the concentrations of dNTPs,primers,Mg2＋ and TaqDNA polymerase.A pair of microsatellite primers from Tachypleus tridentatus was used to amplify microsatellites in Carcinoscorpius rotundicauda.Finally,the optimal SSR-PCR reaction system in Carcinoscorpius rotundicauda was established in 20μL reaction volume,containing 1.5 U TaqDNA polymerases,0.16 mmol/L dNTPs,0.2 μmol/L primers and 2.0 mmol/L Mg2＋.Furthermore,the optimal reaction condition with the optimal concentration（30 ng/μL） of template DNA,optimal annealing temperature（48 ℃）and annealing time（20~25 s）was obtained by gradient PCR reaction.Verification test of the optimized SSR-PCR reaction system and amplificantion procedures showed that this amplification system was stable and practicable.