Hur Deokhwe, Kim Myoung Sug, Song Minsik, Jung Jinwook, Park Heekyung
SeaSun Biomaterials, N517 Deadoek Campus, Pai Chai University, 11-3 Tekeuno 1-ro, Gwanpyeong-dong, Yuseong-gu, Deajeon 305-509 South Korea.
Pathology Division, National Fisheries Research and Development Institute, Busan, 619-902 South Korea.
Biol Proced Online. 2015 Nov 4;17:14. doi: 10.1186/s12575-015-0027-5. eCollection 2015.
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.
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.
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.
探针 - 靶标杂交体的热变性具有高度可重复性，这使得探针熔点分析在检测DNA中的突变、多态性和表观遗传差异方面具有可靠性。为了提高这些检测的分辨率，我们使用了双标记（猝灭剂和荧光）、全碱基的肽核酸（PNA）探针进行基于荧光探针的熔点分析。由于其不带电荷的性质和肽键连接的骨架，PNA探针具有更有利的杂交特性，这使得特异性杂交和部分杂交之间的解链温度有很大差异。
在此，我们表明全碱基双标记PNA是基于荧光探针的熔点分析的合适材料，在完全特异性杂交和部分杂交（包括插入和缺失）的解链温度之间存在很大差异。对于分布狭窄的突变，PNA探针可以用三个探针在单个反应管中有效检测三种突变。此外，我们成功地通过扩增和解链温度信号诊断病毒类似物。最后，仅通过添加γ修饰的PNA探针就可以轻松调节PNA寡聚物的解链温度。
PNA探针在探针设计方面具有提高灵活性的优势，可用于广泛光谱范围内突变检测的各种应用。
