Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis.

Deokhwe Hur,Myoung Sug Kim,Minsik Song,Jinwook Jung,Heekyung Park

doi:10.1186/s12575-015-0027-5

Abstract

BackgroundThermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization.ResultsHere, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe.ConclusionsThe PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-015-0027-5) contains supplementary material, which is available to authorized users.

Highlights

Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA
Real-time PCR assays using fluorescence resonance energy transfer (FRET) probes, molecular beacons, or TaqMan probes have been adapted for continuous mutation detection of amplification products in a closed system
The peptide nucleic acid (PNA) probe designed for detection of three types of variation (SNP, deletion, and insertion) with sequence shift and structural changes that result from small insertion or deletion at the center of the Single Nucleotide Polymorphisms (SNPs) detection In the SNP detection study, hybridization between synthetic DNA oligomer and PNA probe was detected with melting point analysis (Fig. 2a)

Summary

Introduction

Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Real-time PCR assays using fluorescence resonance energy transfer (FRET) probes, molecular beacons, or TaqMan probes have been adapted for continuous mutation detection of amplification products in a closed system. These assays do not effectively distinguish the differences between wild type and mutant types of SNP(s), insertion(s), or deletion(s) because of several handicaps [7].

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Discussion

Conclusion