高GC含量青枯菌aac基因PCR扩增体系的建立与优化

Establishment and optimization of the PCR system for amplification of aac gene from GC-rich Ralstonia solanacearum

通过添加增效剂、正交试验设计优化PCR反应体系、联合采用多种PCR程序等措施,建立并优化了PCR扩增体系,成功地从高GC青枯菌基因组中扩增出了长度为2 434 bp且GC含量高达70.9%的aac基因。PCR反应体系为20μL包括5%DMSO2、.5 mmol/L MgCl2、500μmol/L dNTP、10 pmol/L引物1、.25 UTaq酶、50 ng模板DNA。首先采用热启动PCR:95℃5 min,保持80℃,加入Taq酶;然后采用二步PCR:5个循环包括变性95℃1 min,65℃1 min;最后采用降落PCR:30个循环为95℃1 min,78℃1 min,每个循环降低0.5℃,72℃3 min;补充10个循环为95℃1 min,63℃1 min,72℃3 min;72℃10 min。该试验体系的建立与优化为研究高GC含量生物的基因功能提供了方法。

By introducing different additives and optimizing concentrations with orthogonal test method and combination of different PCR methods, the aac gene was obtained, which is 2 434 bp containing 70.9% of G＋C. The PCR reactions were 20 μL, containing 5% DMSO, 2.5 mmol/L MgCl2 , 500μmol/L dNTPs, 10 pmol/L of each primer, 1.25 unit Taq polymerase and 50 ng genomic DNA. Firstly, Hot-start PCR was performed by denaturing the DNA mixture at 95℃ for 5 min, holding at 80℃ before adding Taq polymerase. Secondly, Two-Step PCR was conducted as followed： 5 cycles at 95℃ for 1 min and 65℃ for 1 min. Thirdly, the touchdown PCR was carried out as followed： 95℃ for 1 min, 78℃ for 1 min with a decrease of 0.5 ℃per cycle, and 72℃ for 3 min. Finally, 10 cycles of 95℃ for 1 min, 63℃ for 1 min, and 72℃ for 3 min were carried out, ended with a 10 min extension at 72℃. The strategy used in this study is very useful for investigation of the genes with high GC content.