| TaqMan实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数 |
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| 引用本文： | 李凯,高宏雷,高立,祁小乐,高玉龙,徐延伟,王笑梅.TaqMan实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数[J].畜牧兽医学报,2011,42(5). |
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| 作者姓名： | 李凯 高宏雷 高立 祁小乐 高玉龙 徐延伟 王笑梅 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所禽传染病研究室兽医生物技术国家重点实验室,哈尔滨,150001 |
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| 基金项目： | 现代农业肉鸡产业技术体系建设项目 |
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| 摘 要： | 以3-磷酸甘油醛脱氢酶为内参基因,建立一种快速检测毕赤酵母基因组中外源基因拷贝数的TaqMan荧光定量PCR方法.通过对反应参数进行优化,建立针对目的基因和内参基因的双标准曲线,并对重组酵母基因组中外源基因和内参基因的拷贝数进行同时定量检测,外源基因与内参基因起始模板拷贝数的比值即为外源基因在重组酵母菌株基因组中的拷贝数.结果表明,2条标准曲线在101～108拷贝·μL-1具有良好的线性关系,以及良好的敏感性和重复性.利用该方法对60株重组酵母菌株进行荧光定量PCR检测,结果筛选到38株单拷贝插入菌株和22株多拷贝插入菌株.本研究所建立的TaqMan探针荧光定量PCR方法方便、高效,可用于重组毕赤酵母基因组中外源基因拷贝数的检测.
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| 关 键 词： | TaqMan荧光定量PCR 毕赤酵母 基因拷贝数 GAP基因 |
Development of a Real-time PCR for Determination of Foreign Gene Copy Number in Genome of Pichia pastoris |
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| LI Kai,GAO Hong-lei,GAO Li,QI Xiao-le,GAO Yu-long,XU Yan-wei,WANG Xiao-mei.Development of a Real-time PCR for Determination of Foreign Gene Copy Number in Genome of Pichia pastoris[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5). |
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| Authors: | LI Kai GAO Hong-lei GAO Li QI Xiao-le GAO Yu-long XU Yan-wei WANG Xiao-mei |
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| Institution: | LI Kai,GAO Hong-lei,GAO Li,QI Xiao-le,GAO Yu-long,XU Yan-wei,WANG Xiao-mei (Division of Avian Infectious Diseases,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China) |
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| Abstract: | A TaqMan real-time PCR assay for rapid determination of the foreign gene copy number in genome of recombinant P.pastoris strains was developed using gp90 gene of reticuloendotheliosis virus as target gene and Glyceraldehydes-3-phosphate dehydrogenase(GAP) gene of P.pastoris as reference gene.The double standard curves of reference gene and gp90 gene were mapped and the genomic DNA of P.pastoris transformants containing gp90 gene was analyzed by real-time PCR,then the gp90/reference ratio in the samples was ... |
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| Keywords: | TaqMan real-time PCR Pichia pastoris gene copy number GAP gene |
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