New morphotypes of condensed DNA microparticles formed in PCR with <Emphasis Type="Italic">KlenTaq</Emphasis> and <Emphasis Type="Italic">Taq</Emphasis> polymerases and plasmid DNA as a template |
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Authors: | V N Danilevich E A Vasilenko E V Pechnikova E V Grishin |
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Institution: | 1.Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,Russian Academy of Sciences,Moscow,Russia;2.Shubnikov Institute of Crystallography,Russian Academy of Sciences,Moscow,Russia |
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Abstract: | The specimens of DNA microparticles formed during PCR amplification of IS-elements ISAfe1 and IST2 by KlenTaq or Taq polymerases and plasmid DNA as a template under varying conditions were investigated by electron microscopy. Microparticle
yield and morphology were found to depend on the level of synthesis of single-stranded DNA fragments during PCR. The conditions
were studied for formation of discs (ellipsoids) several micrometers in diameter and several dozens of nanometers thick, as
well as of microparticles of other morphologies, in the course of PCR with Taq polymerase. The structure of the microparticles produced during an asymmetric PCR, i.e., under conditions of low concentration
of one of the two primers, was investigated. Morphology of the DNA micro- and nanoparticles was found to depend mainly on
the DNA polymerase used in asymmetric PCR. In particular, in the presence of the KlenTaq polymerase, discs or ellipsoids a few dozen nanometers thick were formed, while in the presence of the Taq polymerase, micro- and nanospheres, heterogeneous in size with rugged surfaces, were produced. The effect of Mn2+ cations on DNA microparticle morphology was studied. In the presence of Mn2+, microparticle morphology changed dramatically; in PCR mixtures containing KlenTaq polymerase supplemented with Mn2+, DNA microspheres with fringed surfaces were formed; in the presence of Taq polymerase, microparticles in the form of short, rounded rods were produced. In light of these data, the molecular mechanism
of micro- and nanoparticle formation in the course of PCR is discussed. |
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