Micro- and nanoparticles of condensed DNA Formed in PCR with Taq polymerase and plasmid DNA as a template

Danilevich, V. N.; Vasilenko, E. A.; Pechnikova, Evgeniya V.; Sokolova, Olga S.; Ev, Grishin

doi:10.1134/s0026261711030088

Cited by 4 publications

(9 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Variations in the number of cycles and PCR modes are reported in the Results sec tion. Linear double stranded DNAs, PCR products, as well as plasmid DNAs, were analyzed by electro phoresis in 0.8-1.0% agarose gel [19]. [13].…”

Section: Methodsmentioning

confidence: 99%

“…In the samples under study, aggregates of microspheres 7 μm or more in size were also present (data not shown). Another type of microparticles detected in the samples were the so called three dimensional network struc tures of irregular shape described previously [16,19]. The network size varied in the range from 0.3 to 4 μm.…”

Section: Morphology Of the Microparticles Obtained In Pcr With Klentamentioning

confidence: 99%

“…However, efficient formation of micro and nanoparticles of DNA in the presence of Taq poly merase was recently shown as well [19]. In the work [19], various morphotypes of microparticles formed in the presence of Taq polymerase and plasmid templates were described.…”

mentioning

confidence: 98%

“…Moreover, the presence of single stranded DNA, along with Mg 2+ cations, is essential for DNA condensation dur ing PCR [15,19].…”

mentioning

confidence: 99%

“…In the work [19], various morphotypes of microparticles formed in the presence of Taq polymerase and plasmid templates were described. It was noticed that the efficiency of microparticle formation and their morphology depended upon the rate of nonspecific synthesis of single stranded DNA fragments [19].…”

mentioning

confidence: 99%

See 4 more Smart Citations

New morphotypes of condensed DNA microparticles formed in PCR with KlenTaq and Taq polymerases and plasmid DNA as a template

et al. 2012

Self Cite

View full text Add to dashboard Cite

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 micro particles of other morphologies, in the course of PCR with Taq polymerase. The structure of the microparti cles produced during an asymmetric PCR, i.e., under conditions of low concentration of one of the two prim ers, 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 Mn 2+ cations on DNA microparticle morphology was studied. In the presence of Mn 2+ , microparticle morphology changed dramatically; in PCR mixtures containing KlenTaq polymerase supplemented with Mn 2+ , DNA micro spheres 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 nanopar ticle formation in the course of PCR is discussed.Keywords: polymerase chain reaction (PCR), asymmetric PCR, Taq polymerase, KlenTaq polymerase, Mg 2+ mediated DNA condensation, microparticles and nanoparticles of condensed DNA, effect of Mn 2+ cations, electron microscopy.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Morphology Of the Microparticles Obtained In Pcr With Klentamentioning

confidence: 99%

mentioning

confidence: 98%

“…Moreover, the presence of single stranded DNA, along with Mg 2+ cations, is essential for DNA condensation dur ing PCR [15,19].…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

New morphotypes of condensed DNA microparticles formed in PCR with KlenTaq and Taq polymerases and plasmid DNA as a template

et al. 2012

Self Cite

View full text Add to dashboard Cite

show abstract

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Danilevich¹,

Артемов²,

Smith³

et al. 2013

Journal of Biomolecular Structure and Dynamics

View full text Add to dashboard Cite

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg(2+)-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils - that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg(2+) - mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.

show abstract

New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals

Danilevich

Мачулин

Липкин

et al. 2015

Journal of Biomolecular Structure and Dynamics

View full text Add to dashboard Cite

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg(2+) ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg(2+)-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.

show abstract

Micro- and nanoparticles of condensed DNA Formed in PCR with Taq polymerase and plasmid DNA as a template

Cited by 4 publications

References 14 publications

New morphotypes of condensed DNA microparticles formed in PCR with KlenTaq and Taq polymerases and plasmid DNA as a template

New morphotypes of condensed DNA microparticles formed in PCR with KlenTaq and Taq polymerases and plasmid DNA as a template

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals

Contact Info

Product

Resources

About