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

Biological Procedures Online

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

【 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.

【 图 表 】

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【 参考文献 】

• [1]Altshuler DM, Lander ES, Ambrogio L, Bloom T, Cibulskis K, Fennell TJ et al.. A map of human genome variation from population scale sequencing. Nature. 2010; 467(7319):1061-1073.
• [2]McClellan J, King MC. Genetic heterogeneity in human disease. Cell. 2010; 141(2):210-217.
• [3]Gupta PK, Roy JK, Prasad M. Single nucleotide polymorphisms (SNPs): a new paradigm in molecular marker technology and DNA polymorphism detection with emphasis on their use in plants. Curr Sci. 2001; 80(4):524-535.
• [4]Shi MM. Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem. 2001; 47(2):164-172.
• [5]Hacia JG. Resequencing and mutational analysis using oligonucleotide microarrays. Nat Genet. 1999; 21:42-47.
• [6]Kurg A, Tõnisson N, Georgiou I, Shumaker J, Tollett J, Metspalu A. Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology. Genet Test. 2000; 4(1):1-7.
• [7]Syvanen AC. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet. 2001; 2(12):930-942.
• [8]Porcheddu A, Giacomelli G. Peptide nucleic acids (PNAs), a chemical overview. Curr Med Chem. 2005; 12(22):2561-2599.
• [9]Choi YJ, Kim HJ, Shin HB, Nam HS, Lee SH, Park JS, Baek KA. Evaluation of peptide nucleic acid probe-based real-time PCR for detection of mycobacterium tuberculosis complex and nontuberculous mycobacteria in respiratory specimens. Annals of Laboratory Medicine. 2011; 32(4):257-263.
• [10]Choi YJ, Kim HS, Lee SH, Park JS, Nam HS, Kim HJ, Baek KA. Evaluation of peptide nucleic acid array for the detection of hepatitis B virus mutations associated with antiviral resistance. Arch Virol. 2011; 156(9):1517-1524.
• [11]Kwok PY, Chen X. Detection of single nucleotide polymorphisms. Curr Issues Mol Biol. 2003; 5:43-60.
• [12]Ronaghi M. Pyrosequencing for SNP genotyping. In: Single Nucleotide Polymorphisms. Springer, New York; 2003: p.189-195.
• [13]Sauer S, Gut IG. Genotyping single-nucleotide polymorphisms by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. J Chromatogr B. 2002; 782(1):73-87.
• [14]van der Heiden IP, van der Werf M, Lindemans J, van Schaik RH. Sequencing: not always the “gold standard”. Clin Chem. 2004; 50(1):248-249.
• [15]Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem. 2004; 50(7):1156-1164.
• [16]Huang Q, Liu Z, Liao Y, Chen X, Zhang Y, Li Q. Multiplex fluorescence melting point analysis for mutation detection with dual-labeled, self-quenched probes. PLoS One. 2011; 6(4): Article ID e19206
• [17]Ruskova L, Raclavsky V. The potential of high resolution melting analysis (HRMA) to streamline, facilitate and enrich routine diagnostics in medical microbiology. Biomedical Papers. 2011; 155(3):239-252.
• [18]Sanchez JA, Pierce KE, Rice JE, Wangh LJ. Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc Natl Acad Sci U S A. 2004; 101(7):1933-1938.