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《Journal of Northeast Forestry University》 2006-05
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Optimization of the ISSR-PCR Reaction System for Genetic Diversity Research of Natural Pinus sylvestris var. mongolica

Liu Tong, Hao Yu, Zhou Zhiqiang(Forestry School of Northeast Forestry University, Harbin 150040, P. R. China)  
A set of optimized reaction system and program was set up in terms of the influencing factors of ISSR using genome DNA of Pinus sylvestris var. mongolica (Mongolian Scotch pine) as the template. Results showed that the optimum concentrations of five important components, i.e. template DNA, primer, Mg~ 2+ , dNTP and TaqDNA polymerase were 35ng, 150nmol/L, 1.5mmol/L, 200μmol/L and 1.5U in 20μL reaction mixture, respectively. The reaction program was devised for 5 minutes of predenaturalization at 94℃, 34 cycles of 30 seconds for denaturalization at 94℃, 45 seconds of anneal at 56℃, 2 minutes of extension at 72℃, and 7 minutes of extension at 72℃ in the final cycle. The appropriate anneal temperature was 4℃ higher than Tm of corresponding primer in average, and at least 35 PCR cycles should be conducted to ensure sufficient PCR products. The optimal ISSR reaction system can be applied to the study of genetic differentiation between Mongolian Scotch pine populations.
【Fund】: 黑龙江省青年基金(QC03C23)资助项目。
【CateGory Index】: S791.253
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