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

Naoki Tanigawa,Hodaka Fujii,Toshitsugu Fujita,Richard C Willson

doi:10.1371/journal.pone.0113345

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.

Highlights

Polymerase chain reaction (PCR) is an essential method for molecular biological research
The 39 modifications used for this purpose include dideoxidization [4], nonbase-pairing tails [5,6], and addition of a phosphate group [7]; these approaches are not feasible when used with DNA polymerases that have 39–59 exonuclease activity, because such enzymes can remove the 39 modifications and allow the ODNs or locked nucleic acids (LNAs) to serve as primers
Specific inhibition of PCR amplification using ORNs We reasoned that ORNs could suppress PCR amplification of specific sequences by annealing to the template, and sought to determine whether such ORN-mediated suppression occurs

Summary

Introduction

Polymerase chain reaction (PCR) is an essential method for molecular biological research. The 39 modifications used for this purpose include dideoxidization [4], nonbase-pairing tails [5,6], and addition of a phosphate group [7]; these approaches are not feasible when used with DNA polymerases that have 39–59 exonuclease activity, because such enzymes can remove the 39 modifications and allow the ODNs or LNAs to serve as primers These methods are applicable only when 39–59 exonuclease-deficient DNA polymerases such as Stoffel fragment or Taq polymerases are used [4,5,6,7]. ORNs can be removed by RNases for downstream applications and ORNi-PCR technology should be a useful tool for various applications in molecular biology research and clinical diagnosis

Results and Discussion

Conclusions

Experiments

Materials and Methods