A new strategy to prepare competitive template in quantitative competitive PCR
Competitive PCR is an useful technique in studying gene expression. In this article a new strategy to prepare competitive template, which had homologous primer annealing region and length-different with the target template, was proposed, with IGF-1 gene cDNA as an example. There is an unique restriction endonuclease MspI recognize site in IGF-1 cDNA but thirteens in plasmid pUC-18 backbone. Digested with MspI, the recombinant plasmid pUC-IGF-1 was cut into small fragments with identical sticky ends on their 3and 5terminals. Under the function of T4 DNA ligase these small fragments might be linked with each other randomly. Then the ligation, served as template, was PCR amplified with a pair of primers assigned to IGF-1 cDNA. As a result, bedsides a 160bp IGF-1 cDNA, another DNA fragment about 600bp was successfully amplified. DNA sequencing revealed that this 600bp fragment was formed by an insertion of 440bp into the MspI site of IGF-1 cDNA. Which could be used as competitive template for detecting the concentration of IGF1 transcript in the samples.
competitive PCR template homologous prerare
Bei Sun Aijun Zuo Jingcai Ma Xiaona Cao Dongchun Liang Gang Guo Jingyu Zhang
Institute of Endocrinology/Metabolic Disease Hospital,Tianjin Medical University,Key lab of Hormone and development,Tianjin Ministry,Tianjin 300070,China
国际会议
北京
英文
1-3
2009-06-11（万方平台首次上网日期，不代表论文的发表时间）