期刊论文详细信息
Biological Procedures Online
Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis
Deokhwe Hur1  Myoung Sug Kim2  Minsik Song1  Jinwook Jung1  Heekyung Park1 
[1] SeaSun Biomaterials, N517 Deadoek Campus, Pai Chai University, 11-3 Tekeuno 1-ro, Gwanpyeong-dong, Yuseong-gu 305-509, Deajeon, South Korea
[2] Pathology Division, National Fisheries Research and Development Institute, Busan 619-902, South Korea
关键词: Multiplex;    Probe hybridization;    Mutation;    Genotyping;    Real-time PCR;    Fluorescence probe-based melting point analysis;    Peptide Nucleic Acid (PNA);   
Others  :  1230951
DOI  :  10.1186/s12575-015-0027-5
 received in 2015-09-18, accepted in 2015-10-27,  发布年份 2015
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【 摘 要 】

Background

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. 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.

Results

Here, 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.

Conclusions

The 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.

【 授权许可】

   
2015 Hur et al.

【 预 览 】
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