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[目的]为获得应用于二代测序的高质量16S r DNA V3区PCR产物。[方法]以从人体粪便中提取微生物总DNA为模板,通过梯度PCR和touchdown PCR技术确定了循环条件,并通过调整反应体系中的相关浓度参数以使扩增结果得到优化。[结果]最终确定的循环条件为预变性95℃3 min,变性95℃30 s,退火69℃~62℃每个循环退火温度降0.5℃,退火时间30 s,延伸72℃60 s,共15个循环;变性95℃30 s,退火62℃30 s,延伸72℃60 s,共15个循环,最后72℃延伸5 min。反应体系为:在50μL体系中DNA模板量10~25 ng,pfu酶0.25~0.5 U,正反向引物均为0.06~0.1μmol/L,d NTPs浓度0.2~0.4 mmol/L,Mg2+浓度2~2.5 mmol/L。[结论]梯度PCR与touchdown PCR相结合可快速确定最佳的退火温度以及循环条件,通过调整反应体系中浓度参数可以解决扩增中一些问题。
[Objectives] To obtain a high quality 16S r DNA V3 region PCR product for second generation sequencing. [Method] The total DNA of microorganism was extracted from human excrement as a template, and the cycling conditions were determined by gradient PCR and touchdown PCR. The amplification results were optimized by adjusting the relevant concentration parameters in the reaction system. [Result] The final conditions were as follows: denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 s, annealing at 69 ℃ ~ 62 ℃, annealing temperature decreased by 0.5 ℃ per cycle, annealing time at 30 s, extension at 72 ℃ for 60 s Cycles of denaturation at 95 ° C for 30 s, annealing at 62 ° C for 30 s and extension at 72 ° C for 60 s for a total of 15 cycles with an extension of 5 min at 72 ° C. The reaction system consisted of 10-25 ng DNA template, 0.25-0.5 U pfu enzyme, 0.06-0.1 μmol / L forward and reverse primer, 0.2-0.4 mmol / L dNTPs, 2.5 mmol / L. [Conclusion] The combination of gradient PCR and touchdown PCR can quickly determine the optimal annealing temperature and cycling conditions. Some problems in amplification can be solved by adjusting the concentration parameters in the reaction system.