摘要
【目的】探讨随机引物PCR检测人DNA指纹技术的最优化条件。【方法】应用随机引物PCR技术 ,用一对经筛选的短引物 ,对扩增人DNA指纹的条件进行了优化。【结果】实验结果显示 :DNA模板应新鲜 ,质量浓度在 5 0～ 5 5 0mg/L均有扩增产物。dNTP的浓度 0 2mmol/L最宜。Mg2 + 浓度 5 0mmol/L效果最佳。循环参数以两个三步PCR ,变性温度分别用 94℃、90℃ ,时间各为 30s,退火温度用 43℃、48℃ ,时间分别为 40s、5 0s,延伸温度用 72℃ ,时间分别为 1min、1min 2 0s。【结论】按照优化的实验条件 ,得出的DNA指纹图重复性好 ,个体特异性高 。
To explore the optimal conditions in fingerprinting (APHDF). The human DNA fingerprints were detected by APHDF.A pair of short primers was used for amplification. The experimental conditions including template, Mg 2+ , deoxyribonucleotides, and parameters of cycle, were optimized. The template DNA should be abstracted freshly and the concentration should be ranged from 50～550 mg/L.The best concentration of Mg 2+ was 5 0 mmol/L. The deoxyribonucleotides concentration was optimal at 0 2 mmol/L. The PCR cycling parameters were as follows :The denaturing temperatures,annealing temperatures and extension temperatures were 94 ℃ and 90 ℃ for 30 s, 43 ℃ and 48 ℃ for 40 s or 50 s,and 72 ℃ for 1 min or 80 s, respectively.[Conclusion] The optimal conditions of the experiment are obtained, with good reproducibility and high specificity. Therefore, this method can be widely applied in practice.
出处
《中山医科大学学报》
CSCD
北大核心
2001年第3期231-234,共4页
Academic Journal of Sun Yat-sen University of Medical Sciences
基金
国家自然科学基金资助项目 ( 39370 75 5 )
广东省重点科技攻关项目 ( 1997)
广州市科委资助项目 (JB0 2 97 Z 44 0 2 )
