title: Cooperative primers: 2.5 million-fold improvement in the reduction of nonspecific amplification.
abstract: The increasing need to multiplex nucleic acid reactions presses test designers to the limits of amplification specificity in PCR. Although more than a dozen hot starts have been developed for PCR to reduce primer-dimer formation, none can stop the propagation of primer-dimers once formed. Even a small number of primer-dimers can result in false-negatives and/or false-positives. Herein, we demonstrate a new class of primer technology that greatly reduces primer-dimer propagation, showing successful amplification of 60 template copies with no signal dampening in a background of 150,000,000 primer-dimers. In contrast, normal primers, with or without a hot start, experienced signal dampening with as few as 60 primer-dimers and false-negatives with only 600 primer-dimers. This represents more than a 2.5 million-fold improvement in reduction of nonspecific amplification. We also show how a probe can be incorporated into the cooperative primer, with 2.5 times more signal than conventional fluorescent probes.
authors.affiliation: DNA Logix Inc, Bountiful, Utah. Electronic address: info@dnalogix.net.
authors.forename: Brent C
authors.initials: BC
authors.lastname: Satterfield
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000592:standards*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers