农业生物技术学报2011，Vol.19Issue(6)：1157-1162,6.DOI:10.3969/j.issn.1674-7968.2011.06.024
质粒制备绝对定量PCR标准曲线方法的建立
Establishment of the Plasmid Standard Curve Generation Method for Absolute Quantification PCR
摘要
Abstract
Using plasmid vector to build standard curve for absolute quantification PCR (AQ-PCR) might induce error because of the molecular difference between super-helix double-strand plasmid DNA and linear single-strand cDNA sample. To increase the exactitude of the AQ-PCR, we improved the method by adding two steps during the quantity assay procession of target gene mRNA copy in unknown samples. 1) The plasmid was digested to be linear by restriction enzymes for building standard curve; 2) The unknown cDNA samples were amplified to make a sense strand, followed the fluorescence quantitative PCR cycle with standard plasmid together. The results indicated that 1) The same copy of circular and linear plasmid started AQ-PCR test at the same time,the Ct value of circular plasmid was larger than that of linear plasmid (P0.05); 2) The measured starting copy numbers of cDNA samples amplified once were larger than that of cDNA samples unamplified. The measured results of starting copy number between amplified and unamplified showed that there have 3 of 5 samples were significant differences (P<0.05).Using these improvements, the standard curve of chicken GA TA 5 gene was improved and reflected accurately the amplification of purpose products. Our improved method of using plasmid to build standard curve for AQ-PCR was more feasible, and the measure was more accuracy.关键词
质粒/标准曲线/绝对定量PCRKey words
Plasmid/ Standard Curve/ AQ-PCR引用本文复制引用
李丽,赵成萍,李宏,李武峰,张利环,许冬梅,王金胜,李慧锋..质粒制备绝对定量PCR标准曲线方法的建立[J].农业生物技术学报,2011,19(6):1157-1162,6.基金项目
本研究由中国博士后科学基金资助项目(No.20090451360 （）
No.201003157)、教育部留学回国人员科研启动基金资助项目、山西省回国留学人员科研资助项目(No.2009035)和山西农业大学科技创新基金项目(No.200902)共同资助 （No.2009035）
