| 张争;张杨;徐进;许景升;何礼远;冯洁.高GC含量青枯菌aac基因PCR扩增体系的建立与优化[J].植物保护,2008,34(2):90-93. |
| 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 |
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| DOI： |
| 中文关键词: 高GC PCR 正交试验 |
| 英文关键词:GC-rich polymerase chain reaction(PCR) orthogonal test method |
| 基金项目: |
| 张争;张杨;徐进;许景升;何礼远;冯洁 |
| 中国农业科学院植物保护研究所植物病虫害生物学国家重点试验室 北京100094 |
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| 中文摘要: |
| 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%DMSO、2.5 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。 |
| 英文摘要: |
| 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 MgCl2,500 μmol/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°C 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. |
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