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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Letter Article

Generation and Characterization of Monoclonal Antibodies Against Tth DNA Polymerase and its Application to Hot-Start PCR

Author(s): Yuting Tang, Xiaoyu Chen, Jian Zhang, Jin Wang, Wenhao Hu, Songbai Liu, Zhidan Luo* and Henghao Xu*

Volume 28, Issue 10, 2021

Published on: 05 August, 2021

Page: [1090 - 1098] Pages: 9

DOI: 10.2174/0929866528666210805122117

Price: $65

Abstract

Background: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development.

Objective: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR.

Methods: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/ c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth were incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking the performance of hot-start Tth. Finally, the hot-start Tth was applied to one-step RT-PCR.

Results: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, three monoclonal antibodies were identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies successfully eliminated non-specific amplification in practical applications.

Conclusion: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 had the best overall effect. They possess the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.

Keywords: Tth DNA polymerase, monoclonal antibody, hot-start, PCR, qPCR, inhibitor.

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[1]
Ishmael, F.T.; Stellato, C. Principles and applications of polymerase chain reaction: Basic science for the practicing physician. Ann. Allergy Asthma Immunol., 2008, 101(4), 437-443.
[http://dx.doi.org/10.1016/S1081-1206(10)60323-7] [PMID: 18939735]
[2]
Garcia, J.G.; Ma, S.F. Polymerase chain reaction: A landmark in the history of gene technology. Crit. Care Med., 2005, 33(12)(Suppl.), S429-S432.
[http://dx.doi.org/10.1097/01.CCM.0000186782.93865.00] [PMID: 16340413]
[3]
Rodríguez, M.; Schudel, A.A. Nucleic acid hybridisation and polymerase chain reaction in the diagnosis of infectious animal diseases. Rev. Sci. Tech., 1993, 12(2), 385-423.
[http://dx.doi.org/10.20506/rst.12.2.693] [PMID: 7691273]
[4]
Eom, S.H.; Wang, J.; Steitz, T.A. Structure of Taq polymerase with DNA at the polymerase active site. Nature, 1996, 382(6588), 278-281.
[http://dx.doi.org/10.1038/382278a0] [PMID: 8717047]
[5]
Bhadra, S.; Maranhao, A.C.; Paik, I.; Ellington, A.D. One-enzyme reverse transcription qPCR using Taq DNA polymerase. Biochemistry, 2020, 59(49), 4638-4645.
[http://dx.doi.org/10.1021/acs.biochem.0c00778] [PMID: 33275410]
[6]
Cline, J.; Braman, J.C.; Hogrefe, H.H. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res., 1996, 24(18), 3546-3551.
[http://dx.doi.org/10.1093/nar/24.18.3546] [PMID: 8836181]
[7]
Sankar, P.S.; Citartan, M.; Siti, A.A.; Skryabin, B.V.; Rozhdestvensky, T.S.; Khor, G.H.; Tang, T.H. A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification. Iran. J. Microbiol., 2019, 11(2), 181-186.
[http://dx.doi.org/10.18502/ijm.v11i2.1085] [PMID: 31341574]
[8]
Aye, S.L.; Fujiwara, K.; Ueki, A.; Doi, N. Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication. Biochem. Biophys. Res. Commun., 2018, 499(2), 170-176.
[http://dx.doi.org/10.1016/j.bbrc.2018.03.098] [PMID: 29550479]
[9]
Moreno, R.; Haro, A.; Castellanos, A.; Berenguer, J. High-level overproduction of his-tagged Tth DNA polymerase in Thermus thermophilus. Appl. Environ. Microbiol., 2005, 71(1), 591-593.
[http://dx.doi.org/10.1128/AEM.71.1.591-593.2005] [PMID: 15640243]
[10]
Rüttimann, C.; Cotorás, M.; Zaldívar, J.; Vicuña, R. DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8. Eur. J. Biochem., 1985, 149(1), 41-46.
[http://dx.doi.org/10.1111/j.1432-1033.1985.tb08890.x] [PMID: 3996403]
[11]
Nolan, T.; Hands, R.E.; Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc., 2006, 1(3), 1559-1582.
[http://dx.doi.org/10.1038/nprot.2006.236] [PMID: 17406449]
[12]
Glukhov, A.I.; Grebennikova, T.V.; Kiselev, V.I.; Severin, E.S. Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction for detecting CD4 receptor mRNA. Mol. Biol. (Mosk.), 1995, 29(4), 942-949.
[PMID: 7476959]
[13]
Grebennikova, T.V.; Glukhov, A.I.; Chistiakova, L.G.; Kiselev, V.I.; Severin, E.S. Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1). Mol. Biol. (Mosk.), 1995, 29(4), 930-941.
[PMID: 7476958]
[14]
Moser, M.J.; DiFrancesco, R.A.; Gowda, K.; Klingele, A.J.; Sugar, D.R.; Stocki, S.; Mead, D.A.; Schoenfeld, T.W. Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme. PLoS One, 2012, 7(6), e38371.
[http://dx.doi.org/10.1371/journal.pone.0038371] [PMID: 22675552]
[15]
Chiocchia, G.; Smith, K.A. Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase. Biotechniq., 1997, 22(2), 312-314, 316, 318.
[http://dx.doi.org/10.2144/97222st04] [PMID: 9043704]
[16]
Furutani, S.; Nagai, H.; Takamura, Y.; Aoyama, Y.; Kubo, I. Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method. Analyst (Lond.), 2012, 137(13), 2951-2957.
[http://dx.doi.org/10.1039/c2an15866c] [PMID: 22234623]
[17]
Myers, T.W.; Gelfand, D.H. Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry, 1991, 30(31), 7661-7666.
[http://dx.doi.org/10.1021/bi00245a001] [PMID: 1714296]
[18]
Kim, J.; Jung, S.; Kim, M.Y.; Kim, B.K.; Kwon, S.H.; Kim, S.K. Thermo-responsive polymer capsules in real-time one-step RT-PCR for highly multiplex RNA analysis. Adv. Healthc. Mater., 2020, 9(7), e1900790.
[http://dx.doi.org/10.1002/adhm.201900790] [PMID: 32134572]
[19]
Wiedbrauk, D.L.; Werner, J.C.; Drevon, A.M. Inhibition of PCR by aqueous and vitreous fluids. J. Clin. Microbiol., 1995, 33(10), 2643-2646.
[http://dx.doi.org/10.1128/jcm.33.10.2643-2646.1995] [PMID: 8567898]
[20]
Wang, L.F.; Radkowski, M.; Vargas, H.; Rakela, J.; Laskus, T. Amplification and fusion of long fragments of hepatitis C virus genome. J. Virol. Methods, 1997, 68(2), 217-223.
[http://dx.doi.org/10.1016/S0166-0934(97)00132-8] [PMID: 9389412]
[21]
Hanaki, K.; Odawara, T.; Muramatsu, T.; Kuchino, Y.; Masuda, M.; Yamamoto, K.; Nozaki, C.; Mizuno, K.; Yoshikura, H. Primer/template-independent synthesis of poly d(A-T) by Taq polymerase. Biochem. Biophys. Res. Commun., 1997, 238(1), 113-118.
[http://dx.doi.org/10.1006/bbrc.1997.7197] [PMID: 9299462]
[22]
Ignatov, K.B.; Kramarov, V.M.; Uznadze, O.L.; Miroshnikov, A.I. DNA polymerase mediated amplification of DNA fragments using primers with mismatches in the 3′-region. Bioorg. Khim., 1997, 23(10), 817-822.
[PMID: 9490618]
[23]
Green, M.R.; Sambrook, J. Hot start polymerase chain reaction (PCR). Cold Spring Harb. Protoc., 2018, 2018(5)
[http://dx.doi.org/10.1101/pdb.prot095125] [PMID: 29717052]
[24]
Ward, E.S.; Güssow, D.; Griffiths, A.D.; Jones, P.T.; Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 1989, 341(6242), 544-546.
[http://dx.doi.org/10.1038/341544a0] [PMID: 2677748]
[25]
D’Aquila, R.T.; Bechtel, L.J.; Videler, J.A.; Eron, J.J.; Gorczyca, P.; Kaplan, J.C. Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucleic Acids Res., 1991, 19(13), 3749.
[http://dx.doi.org/10.1093/nar/19.13.3749] [PMID: 1852616]
[26]
Rimseliene, R. Antibodies that bind Thermophilc DNA polymerase. U.S. Patent 0204454A1, 2017.
[27]
Sharkey, D.J.; Scalice, E.R.; Christy, K.G., Jr; Atwood, S.M.; Daiss, J.L. Antibodies as thermolabile switches: High temperature triggering for the polymerase chain reaction. Biotechnol. (N. Y.), 1994, 12(5), 506-509.
[http://dx.doi.org/10.1038/nbt0594-506] [PMID: 7764710]
[28]
Kellogg, D.E.; Rybalkin, I.; Chen, S.; Mukhamedova, N.; Vlasik, T.; Siebert, P.D.; Chenchik, A. TaqStart antibody: “Hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniq., 1994, 16(6), 1134-1137.
[PMID: 8074881]
[29]
Hébert, B.; Bergeron, J.; Potworowski, E.F.; Tijssen, P. Increased PCR sensitivity by using paraffin wax as a reaction mix overlay. Mol. Cell. Probes, 1993, 7(3), 249-252.
[http://dx.doi.org/10.1006/mcpr.1993.1036] [PMID: 8366871]
[30]
Kaijalainen, S.; Karhunen, P.J.; Lalu, K.; Lindström, K. An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose. Nucleic Acids Res., 1993, 21(12), 2959-2960.
[http://dx.doi.org/10.1093/nar/21.12.2959] [PMID: 8332517]
[31]
Chou, Q.; Russell, M.; Birch, D.E.; Raymond, J.; Bloch, W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res., 1992, 20(7), 1717-1723.
[http://dx.doi.org/10.1093/nar/20.7.1717] [PMID: 1579465]
[32]
Ariel, L.; Anne, V. Thermally reversible inactivation of Taq polymerase in an organic solvent for application in Hot Start PCR. Enzyme Microb. Technol., 2005, 36(7), 947-952.
[http://dx.doi.org/10.1016/j.enzmictec.2005.01.019]
[33]
Dang, C.; Jayasena, S.D. Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. J. Mol. Biol., 1996, 264(2), 268-278.
[http://dx.doi.org/10.1006/jmbi.1996.0640] [PMID: 8951376]
[34]
Noma, T.; Sode, K.; Ikebukuro, K. Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm. Biotechnol. Lett., 2006, 28(23), 1939-1944.
[http://dx.doi.org/10.1007/s10529-006-9178-4] [PMID: 16988782]
[35]
Dahiya, R.; Deng, G.; Chen, K.; Haughney, P.C.; Cunha, G.R.; Narayan, P. Terms and techniques: New approach to hot-start polymerase chain reaction using Taq DNA polymerase antibody. Urol. Oncol., 1995, 1(1), 42-46.
[http://dx.doi.org/10.1016/1078-1439(95)00001-X] [PMID: 21224089]
[36]
Paul, N.; Shum, J.; Le, T. Hot start PCR. Methods Mol. Biol., 2010, 630, 301-318.
[http://dx.doi.org/10.1007/978-1-60761-629-0_19] [PMID: 20301005]
[37]
Obradovic, D.; Kevresan, S. Optimization of PCR in application of hot start Taq DNA polymerase for detection of Erwinia amylovora with primers FER1-F and FER1-R. Mikrobiologiia, 2010, 79(6), 819-823.
[PMID: 21446634]
[38]
Chen, X.Y.; Zhang, J.; Zhang, X.Y.; Tang, Y.T.; Shao, Y.C.; Luo, Z.D.; Lu, C. A rapid and accurate method for Tth DNA polymerase activity assay. Biotechnol. Bulletin, , 2021., 37(5), 281-286.
[39]
Cai, D.; Behrmann, O.; Hufert, F.; Dame, G.; Urban, G. Direct DNA and RNA detection from large volumes of whole human blood. Sci. Rep., 2018, 8(1), 3410.
[http://dx.doi.org/10.1038/s41598-018-21224-0] [PMID: 29467420]
[40]
Terpe, K. Overview of thermostable DNA polymerases for classical PCR applications: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol., 2013, 97(24), 10243-10254.
[http://dx.doi.org/10.1007/s00253-013-5290-2] [PMID: 24177730]
[41]
Poddar, S.K.; Sawyer, M.H.; Connor, J.D. Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus. J. Med. Microbiol., 1998, 47(12), 1131-1135.
[http://dx.doi.org/10.1099/00222615-47-12-1131] [PMID: 9856650]
[42]
Danilevich, V.N.; Vasilenko, E.A.; Pechnikova, E.V.; Grishin, E.V. New morphotypes of condensed DNA microparticles formed in PCR with KlenTaq- and Taq-polymerases and with plasmid DNAs as templates. Mikrobiologiia, 2012, 81(1), 126-137.
[http://dx.doi.org/10.1134/S002626171106004X] [PMID: 22629690]

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