拟南芥SSR检测体系的优化

Optimization of SSR detection system for Arabidopsis thaliana

以拟南芥(Arabidopsis thaliana)幼苗为实验材料,采用单因素筛选法及L16(45)正交实验方法,对简单序列重复(SSR)技术中聚合酶链式反应(PCR)组分、扩增程序、电泳检测等环节进行优化。优化25μl反应体系为:1×PCR Buffer、20ng模板DNA、1.5mmol.L-1Mg2+、0.3μmol·L-1引物、150μmol·L-1dNTPs和1.0U Taq DNA聚合酶。扩增程序为:94℃预变性5min,94℃变性30s,57℃退火30s,72℃延伸45s,共30个循环,72℃延伸10min。用非变性聚丙烯酰胺凝胶(EB染色)电泳检测并取得较好效果。利用该体系进行扩增,所得谱带清晰、稳定、非特异性带少。

Taking Arabidopsis thaliana as test material and by the methods of single factor selection and L16（4^5） orthogonal design,the amplification component,program,and electrophoresis detection in polymerase chain reaction （PCR） in simple sequence repeat （SSR）-detection system were optimized. The optimized 25 μl reaction system contained 1×PCR Buffer,20 ng DNA template,1.5 mmol·L^-1 of Mg^2＋,0.3 μmol·L-1 of primers,150 μmol·L^-1 of dNTPs,and 1.0 U of Taq DNA polymerase. The PCR amplification procedures consisted of an initial denaturing for 5 min at 94 ℃,followed by 30 cycles of denaturation for 30 s at 94 ℃,annealing for 30 s at 57 ℃,and an extension for 45 s at 72 ℃,with an additional extension period of 10 min at 72 ℃. By using non-denaturing polyacrylamide gel electrophoresis with ethidium bromide （EB） staining,the amplification products bands of SSR were easy to be identified. In the SSR system,amplification bands were clear and stable,and there were fewer non-target bands.