Abstract
Earlier, we discovered that, along with linear DNA fragments, nano- and microparticles of DNA and their aggregates are formed in the PCR with yeast genomic DNA used as a template and gene-specific or partially complementary primers. The size of the microparticles (microspheres) varied in the range of 0.5 to 3–4 μm. Only thermostable KlenTaq polymerase but not Taq polymerase could effectively generate microspheres. In this work, we demonstrate that KlenTaq polymerase can produce microspheres of variable size (1 to 7 μm in diameter) if genomic DNA of the bacterium Acidithiobacillus ferrooxidans and partially complementary primers are present in the PCR mixture. Conditions for generation of DNA microparticles in PCR with Taq-polymerase and bacterial genomic DNA as template were also elaborated. It was also found that mainly large microspheres of up to 7 μm accumulated in PCR with plasmid DNAs used as templates and gene-specific primers in the presence of KlenTaq polymerase or mixtures of KlenTaq and Pfu polymerases. Besides, small aggregates, as well as linear branched structures and three-dimensional conglomerates of fused microspheres, were also revealed in the PCR mixtures. UV absorption spectra of native DNA microspheres and microspheres that had undergone heating at 93°C were registered. The key role of Mg2+ cations in the formation and stabilization of the microsphere structure was established.
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Danilevich, V.N., Petrovskaya, L.E., and Grishin, E.V., DNA Nano- and Microparticles: New Products of Polymerase Chain Reaction, Dokl. Akad. Nauk, 2008, vol. 421, no. 1, pp. 119–122 [Dokl. Biochem. Biophys. (Engl. Transl.), 2008, vol. 421, nos. 1–6, pp. 168–170].
Danilevich, V.N., Barinova, E.S., and Grishin, E.V., Microspheres of Condensed DNA Formed in the Process of Polymerase Chain Reaction, Bioorg. Khim., 2009, vol. 35, no. 2, pp. 226–238.
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A., Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase, Science, 1988, vol. 239, pp. 487–491.
Lawyer, F.C., Stoffe, S., Saiki, R.K., Myambo, K., Drummond, R., and Gelfand, D.H., Isolation, Characterization, and Expression in Escherichia coli of the DNA Polymerase Gene from Thermus aquaticus, J. Biol. Chem., 1989, vol. 264, pp. 6427–6437.
Fang, Y. and Hoh, J.H., Surface-Directed DNA Condensation in the Absence of Soluble Multivalent Cations, Nucleic Acids Res., 1998, vol. 26, pp. 588–593.
Bloomfiel, V.A., Condensation of DNA by Multivalent Cations: Considerations on Mechanism, Biopolymers, 1991, vol. 31, pp. 1471–1481.
Skuridin, S.G., Kadykov, V.A., Shashkov, V.S., Evdokimov, Yu.M., and Varshavskii, Ya.M., Production of Compact DNA Forms in Solution upon the Interaction with Spermidine, Mol. Biol., 1978, vol. 12, no. 2, pp. 413–420.
Lortkipanidze, G.B., Evdokimov, Yu.M., Kadykov, V.A., and Varshavskii, Ya.M., Compact Particles of Double-Stranded Polyribonucleotides. I. Conditions of Formation of Optically Active Compact Particles from Double-Stranded RNA, Mol. Biol., 1980, vol. 14, no. 6, pp. 1378–1386.
Teif, V.B., Ligand-Induced DNA Condensation: Choosing the Model, Biophys. J., 2005, vol. 89, pp. 2574–2587.
Kornyshev, A.A. and Leikin, S., Electrostatic Interaction between Helical Macromolecules in Dense Aggregates: An Impetus for DNA Poly- and Meso-Morphism, Proc. Natl. Acad. Sci. USA, 1998, vol. 5, pp. 13579–13584.
Ma, C. and Bloomfield, V.A., Condensation of Super-coiled DNA Induced by MnCl2, Biophys. J., 1994, vol. 67, pp. 1678–1681.
Wu, G.Y. and Wu, C.H., Receptor-Mediated Gene Delivery and Expression In Vivo, J. Biol. Chem., 1988, vol. 263, pp. 14621–14624.
Shapiro, J.T., Leng, M., and Felsenfeld, G., Deoxyribo-nucleic Acid-Polylysine Complexes. Structure and Nucleotide Specificity, Biochemistry, 1969, vol. 8, pp. 3219–3232.
Niidome, T., Ohmori, N., Ichinose, A., Wada, A., Mihara, H., Hirayama, T., and Aoyagi, H., Binding of Cationic Alpha-Helical Peptides to Plasmid DNA and Their Gene Transfer Abilities Into Cells, J. Biol. Chem., 1997, vol. 272, pp. 15307–15312.
Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M., Lipofection: a Highly Efficient, Lipid-Mediated DNA-Transfection Procedure, Proc. Natl. Acad. Sci. USA, 1987, vol. 84, pp. 7413–7417.
Kondrat’eva, T.F., Danilevich, V.N., Ageeva, S.N., and Karavaiko, G.I., Identification of IS Elements in Acidithiobacillus ferrooxidans Strains Grown in a Medium with Ferrous Iron or Adapted to Elemental Sulfur, Arch. Microbiol., 2005, vol. 183, pp. 401–410.
Fields, S. and Song, O., A Novel Genetic System To Detect Protein-Protein Interactions, Nature, 1989, vol. 340, pp. 245–246.
Maniatis, T., Fritsch, E.F., and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Lab. Press, 1989, pp. 17–38.
Permogorov, V.I., Tyaglov, B.V., Bogush, V.G., and Minaev, V.E., Determination of Light Dispersion during Melting of Phages T7 and Sd, Mol. Biol., 1977, vol. 11, no. 1, pp. 134–138.
Laemmli, U.K., Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature, 1970, vol. 227, pp. 680–685.
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Original Russian Text © V.N. Danilevich, E.V. Grishin, 2009, published in Mikrobiologiya, 2009, Vol. 78, No. 3, pp. 369–380.
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Danilevich, V.N., Grishin, E.V. Characteristics of microspheres formed in PCR with bacterial genomic DNA or plasmid DNA as templates. Microbiology 78, 328–338 (2009). https://doi.org/10.1134/S0026261709030114
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DOI: https://doi.org/10.1134/S0026261709030114