Abstract
This chapter introduces neutralized DNA (nDNA) as a novel design for the primers of PCR and RT-PCR by methylating phosphate groups of some oligonucleotides in their structures. It starts with an introduction of the nDNA which possesses an electrically chimeric neutral backbone as well as the proposed standards in designing nDNA as a novel primer for PCR and RT-PCR , concluded from various experimental results presented afterward. The primary content comprises empirical data from PCR to compare nDNA and unmodified DNA as primers in terms of ability to distinguish and amplify mismatch templates, activities of polymerase enzymes, melting temperature of double-stranded sequences, and the trials and discussions on various modified positions of the nDNA primers. In summary, nDNA exhibited outstanding performance as a primer for PCR and RT-PCR , compared to unmodified DNA, and is expected to be expanded in diverse applications which require enhanced specificity.
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Kawane K, Motani K, Nagata S (2014) DNA degradation and its defects. Cold Spring Harb Perspect Biol 6:a016394. https://doi.org/10.1101/cshperspect.a016394
Obika S, Nanbu D, Hari Y, Morio K, In Y, Ishida T, Imanishi T (1997) Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering. Tetrahedron Lett 38(50):8735–8738. https://doi.org/10.1016/S0040-4039(97)10322-7
Levin JD, Fiala D, Samala MF, Kahn JD, Peterson RJ (2006) Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers. Nucleic Acids Res 34:e142. https://doi.org/10.1093/nar/gkl756
Mouritzen P, Nielsen AT, Pfundheller HM, Choleva Y, Kongsbak L, Moller S (2003) Single nucleotide polymorphism genotyping using locked nucleic acid (LNA™). Expert Rev Mol Diagn 3:27–38. https://doi.org/10.1586/14737159.3.1.27
Egholm M, Buchardt O, Nielsen PE, Berg RH (1992) Peptide nucleic acids (PNA). Oligonucleotide analogs with an achiral peptide backbone. J Am Chem Soc 114:1895–1897
Wang J, Palecek E, Nielsen PE, Rivas G, Cai X, Shiraishi H, Dontha N, Luo D, Farias PAM (1996) Peptide nucleic acid probes for sequence-specific DNA biosensors. J Am Chem Soc 118:7667–7670. https://doi.org/10.1021/ja9608050
Ray A, Norden B (2000) Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future. FASEB J 14:1041–1060. https://doi.org/10.1096/fasebj.14.9.1041
Chen WY, Chen HC, Yang YS, Huang CJ, Chan HWH, Hu WP (2013) Improved DNA detection by utilizing electrically neutral DNA probe in field-effect transistor measurements as evidenced by surface plasmon resonance imaging. Biosens Bioelectron 41:795–801. https://doi.org/10.1016/j.bios.2012.10.010
Hu WP, Tsai CC, Yang YS, HWH C, Chen WY (2018) Synergetic improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing neutralized DNA as probe. Sci Rep 8:12598. https://doi.org/10.1038/s41598-018-30996-4
Kuo TC, Wu MW, Lin WC, Matulis D, Yang YS, Li SY, Chen WY (2020) Reduction of interstrand charge repulsion of DNA duplexes by salts and by neutral phosphotriesters—contrary effects for harnessing duplex formation. J Taiwan Ins Chem Eng 110:1–7. https://doi.org/10.1016/j.jtice.2020.02.023
Li TL, Wu MW, Lin WC, Lai CH, Chang YH, Su LJ, Chen WY (2019) Designed phosphate-methylated oligonucleotides as PCR primers for SNP discrimination. Anal Bioanal Chem 411:3871–3880. https://doi.org/10.1007/s00216-019-01865-4
Chen WY, Matulis D, Hu WP, Lai YF, Wang WH (2020) Studies of the interactions mechanism between DNA and silica surfaces by isothermal titration calorimetry. J Taiwan Ins Chem Eng 116:62–66. https://doi.org/10.1016/j.jtice.2020.11.019
Coenen AJJM, Henckens LHG, Mengerink Y, van der Wal S, Quaedflieg PJLM, Koole LH, Meijer EM (1992) Optimization of the separation of the Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides. J Chromatogr 596:59–66. https://doi.org/10.1016/0021-9673(92)80202-6
Roush S, Slack FJ (2008) The let-7 family of microRNAs. Trends Cell Biol 18:505–516. https://doi.org/10.1016/j.tcb.2008.07.007
Vinogradova OA, Pyshnyi DV (2010) Selectivity of enzymatic conversion of oligonucleotide probes during nucleotide polymorphism analysis of DNA. Acta Nat 2:36–53
Hong CY, Chen WY "Studies of improving detection specificity of single nucleotide variation and miRNAs by phosphate methylated oligoDNA primers" Master Thesis of Department of Chemical and Materials Engineering, National Central University, July, 2021
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Chang, YH., Wu, MW., Chen, YJ., Vu, CA., Hong, CY., Chen, WY. (2022). Phosphate-Methylated Oligonucleotides as a Novel Primer for PCR and RT-PCR. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 2392. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1799-1_18
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DOI: https://doi.org/10.1007/978-1-0716-1799-1_18
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