PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR

Abstract

A rapid and reliable method is described for preparing competitive DNA fragments for quantitative PCR. Synthetic DNAs complementary to previously established PCR primers are ligated together with the primers to both ends of a generic DNA fragment whose length differs from the natural target gene PCR product. After a short ligation step, the properly constructed ligation products (i.e., those that have the correct primer templates on opposite sides of the generic DNA fragment) are preferentially amplified by PCR. The generation of competitive PCR fragments, MIMICS, can be completed in a single day. To perform quantitative PCR, known quantities of PCR MIMICS are spiked into PCR amplification reactions containing the experimental cDNA samples. A visual or radioactive comparison of the PCR products can then be used to determine the initial quantity of target gene. We show that competitive PCR MIMICS can be used to accurately measure small changes in mRNA levels.