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 Mspl recognize site in IGF-1 cDNA but thirteens in plasmid pUC-18 backbone. Digested with Mspl, the recombinant plasmid pUC-IGF-1 was cut into small fragments with identical sticky ends on their 3'and 5'terminals. 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 160 bp IGF-1 cDNA, another DNA fragment about 600 bp was successfully amplified. DNA sequencing revealed that this 600 bp fragment was formed by an insertion of 440 bp into the Mspl site of IGF-1 cDNA. Which could be used as competitive template for detecting the concentration of IGF1 transcript in the samples.