Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site.

ABSTRACT: Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by

SUBMITTER: Watzinger F 

PROVIDER: S-EPMC55722 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature