| 邻近多位点基因突变的组合大引物PCR策略(英文) |
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| 引用本文： | 赵广荣,梁玲玲,刘春杰,祝琳琪.邻近多位点基因突变的组合大引物PCR策略(英文)[J].中国生物工程杂志,2010,30(7). |
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| 作者姓名： | 赵广荣 梁玲玲 刘春杰 祝琳琪 |
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| 作者单位： | 教育部系统生物工程重点实验室,天津大学化工学院制药工程系,天津,300072 |
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| 基金项目： | 高等学校博士学科点专项科研基金，天津市自然科学基金 |
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| 摘 要： | 报道了一种新的组合多位点突变策略,通过单管中三阶段聚合酶链式反应(PCR)得以实现。在第一阶段,PCR扩增出多位点突变大引物,然后在第二阶段延伸大引物,在第三阶段获得全长突变基因序列。基于退火温度与热循环参数的组合大引物反应的优化,三个阶段中退火温度差异小(低于10°C),成功扩增出多位点突变基因序列和比邻突变序列。是一种简单、高效的多位点突变方法。
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| 关 键 词： | 多位点突变 大引物PCR 基因工程 组合三阶段PCR反应 人干扰素α |
A Combinatorial Megaprimer PCR Strategy for Adjacent Multi-site-directed Gene Mutagenesis |
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| ZHAO Guang-rong,LIANG Ling-ling,LIU Chun-jie,ZHU Lin-qi.A Combinatorial Megaprimer PCR Strategy for Adjacent Multi-site-directed Gene Mutagenesis[J].China Biotechnology,2010,30(7). |
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| Authors: | ZHAO Guang-rong LIANG Ling-ling LIU Chun-jie ZHU Lin-qi |
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| Abstract: | A novel combinatorial multi-site directed mutagenesis strategy was developed,which performed in three-stage PCR in one tube.The multi-site mutagenic megaprimer is amplified in the first stage PCR,then the megaprimer are extended in the second stage PCR,and the full length mutant sequence is generated in the third stage PCR.Based on combinatorial megaprimer reactions of the annealing temperatures and thermo-cycles,the multi-site directed and adjacent mutagenic DNA fragments were successfully amplified with the small annealing temperature difference (lesser than 10℃) between three stage PCRs.This method is simple and high efficient. |
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| Keywords: | Multi-site mutagenesis Megaprimer PCR Genetic engineering Combinatorial three-stage PCR reaction Human interferon α |
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