Cooperative primers: 2.5 million-fold improvement in the reduction of nonspecific amplification

J Mol Diagn. 2014 Mar;16(2):163-73. doi: 10.1016/j.jmoldx.2013.10.004. Epub 2013 Dec 25.


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.

MeSH terms

  • DNA Primers / chemistry
  • DNA Primers / standards*
  • Humans
  • Multiplex Polymerase Chain Reaction / methods*
  • Multiplex Polymerase Chain Reaction / standards*
  • Reproducibility of Results
  • Sensitivity and Specificity


  • DNA Primers