A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gen...A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.展开更多
Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished f...Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.展开更多
The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatiti...The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.展开更多
本试验旨在制备用于小尾寒羊瘦素长型受体(leptin receptor long form)基因实时荧光定量PCR的标准品质粒和标准曲线。提取初情期前小尾寒羊下丘脑弓状核组织总RNA,进行反转录合成第1链cDNA,然后进行PCR和目的基因片段的回收;通过T-A克...本试验旨在制备用于小尾寒羊瘦素长型受体(leptin receptor long form)基因实时荧光定量PCR的标准品质粒和标准曲线。提取初情期前小尾寒羊下丘脑弓状核组织总RNA,进行反转录合成第1链cDNA,然后进行PCR和目的基因片段的回收;通过T-A克隆将该基因片段插入pMD18-T载体中,构建回收产物的重组质粒;大量提取质粒,定量后进行标准曲线的制作和实时荧光定量PCR。重组质粒经PCR扩增和测序,结果表明,瘦素长型受体基因已成功克隆。提示,所构建的瘦素长型受体基因实时荧光定量标准曲线线性关系好,灵敏度和特异性高,准确可靠,此方法可作为实时荧光定量PCR检测瘦素长型受体基因的标准方法。展开更多
基金Sponsored by the Shanghai Academy of Agricultural Science and Shanghai Science Committee (No .003912076)
文摘A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.
文摘Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.
文摘The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.
文摘本试验旨在制备用于小尾寒羊瘦素长型受体(leptin receptor long form)基因实时荧光定量PCR的标准品质粒和标准曲线。提取初情期前小尾寒羊下丘脑弓状核组织总RNA,进行反转录合成第1链cDNA,然后进行PCR和目的基因片段的回收;通过T-A克隆将该基因片段插入pMD18-T载体中,构建回收产物的重组质粒;大量提取质粒,定量后进行标准曲线的制作和实时荧光定量PCR。重组质粒经PCR扩增和测序,结果表明,瘦素长型受体基因已成功克隆。提示,所构建的瘦素长型受体基因实时荧光定量标准曲线线性关系好,灵敏度和特异性高,准确可靠,此方法可作为实时荧光定量PCR检测瘦素长型受体基因的标准方法。
