Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

Yasumasa Kimura,Takahiro Soma,Diane Delobel,Michiel J L De Hoon,Yoshihide Hayashizaki,Naoko Kasahara,Takeshi Hanami,Matthias Harbers,Yuki Tanaka,Kengo Usui,Ruslan Kalendar

doi:10.1371/journal.pone.0146950

Abstract

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

Highlights

Many PCR applications in research and medical diagnostics monitor amplification reactions in real-time to directly detect the amplification product or to obtain quantitative results
While very good programs are available for the design of PCR primers, we saw a need for a better tool for selecting internal probes for quantitative real-time PCR (qPCR) experiments
The tool should be suitable for developing internal probes for genotypic assays that are often conducted by melting curve analysis of the PCR product

Summary

Introduction

Many PCR applications in research and medical diagnostics monitor amplification reactions in real-time to directly detect the amplification product or to obtain quantitative results. This is achieved by adding an internal probe to the reaction that hybridizes to the amplification product in a sequence-dependent manner [1]. Two separate oligonucleotides have been used as so-called HybProbes [6], where one oligonucleotide carries a donor and the other oligonucleotide carries an acceptor dye All those internal probes can only be designed in the context of the primers that are used in the amplification reaction. Many laboratories are using commercial applications offered by different providers; for a list of software tools refer to [13]

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Conclusion