Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR): a simple method for suppressing PCR amplification of specific DNA sequences using ORNs

PLoS One. 2014 Nov 18;9(11):e113345. doi: 10.1371/journal.pone.0113345. eCollection 2014.

Abstract

Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effective at very low concentrations, with IC50 values for ORN-mediated suppression on the order of 10 nM. DNA polymerases that retain 3'-5' exonuclease activity, such as KOD and Pfu polymerases, but not those that retain 5'-3' exonuclease activity, such as Taq polymerase, could be used for ORN-mediated suppression. ORN interference-PCR (ORNi-PCR) technology should be a useful tool for both molecular biology research and clinical diagnosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA / genetics
  • DNA / metabolism
  • DNA Primers / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Models, Genetic*
  • Oligoribonucleotides / genetics*
  • Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Taq Polymerase / metabolism
  • Templates, Genetic

Substances

  • DNA Primers
  • Oligoribonucleotides
  • DNA
  • Pfu DNA polymerase
  • Taq Polymerase
  • DNA-Directed DNA Polymerase

Grants and funding

This study was supported by Research Institute for Microbial Diseases, Osaka University (TF, HF). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.