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An electron microscopy study of large-sized DNA microparticles produced in PCR with different gene-specific primers and plasmid DNAs is described. DNA microspheres of two distinct types were revealed in the all studied samples, namely smooth moderately electron-dense microspheres, and highly electron-dense particles with large thorns and offshoots. Singular microspheres have the average diameter of 1 mum, and their aggregates were up to 3 mum in dimensions. In addition, rare so-called three-dimensional net-like structures with various size (up to several micrometers) were observed. They consisted of different amounts of DNA nanoparticles, having the special compact topology. In some studied samples the discs (nanodiscs) of several dozens nm in thickness and up to 3 mum in diameter were revealed. It was shown that the quantity of net-like structures and nanodiscs sharply increases in asymmetric PCR. We also observed DNA nanowires of different length and thickness, nanodots, nanoparticles in the form of shits of paper as well as electron-dense spherical nanoparticles of big size. Aqueous suspensions of DNA microparticles were heated at 94 degrees C for 5 min and analyzed by electron microscopy. It was shown that microspheres in heated suspensions underwent partial melting; they lost a part of DNA, therefore details of their structure (ultrastructure) can be recognized. At the some time numerous tangles of nanowires appeared. Molecular mechanisms of the DNA micro- and nanoparticles formation are discussed.

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3-4 microm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg2+ cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.

The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.

A simple and rapid procedure for the preparation of yeast and fungal DNA samples useful in PCR amplification was developed. The DNA was purified from proteins, lipids, polysaccharides, and other impurities by their high-temperature extraction (in a boiling water bath) with buffer solutions containing chaotropic salts. Under these conditions, yeast and fungal cell envelopes remain unbroken and retain the original DNA and RNA that could be used for direct PCR amplification. We called the proposed PCR technique as the PCR using DNA-containing cell envelopes.

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 Mn cations on DNA microparticle morphology was studied. In the presence of Mn, microparticle morphology changed dramatically; in PCR mixtures containing KlenTaq polymerase supplemented with Mn, 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. [ABSTRACT FROM AUTHOR]

Suspensions of microparticles produced in a polymerase chain reaction on plasmid templates using different primers have been studied by electron microscopy. In all of the samples, microspheres of two types have been detected: of a spherical or ellipsoid form (smooth, without thorns) and of an ellipsoid form with big thorns and/or outgrowths; the diameter of the microspheres varies from 1 to 3 μm. Along with microspheres, so-called three-dimensional network structures of various shapes formed by three-dimensional nanoparticles of a particular topology have been observed. In some samples, discs a few microns in diameter and several dozen nanometers in thickness have been detected. It has been shown that the quantity of netshaped structures and discs sharply increases during asymmetric PCR. In addition, DNA nanofilaments (nanowires), electron-dense point particles (nanodots), electron-translucent nanoparticles in the form of rags, and large electron-dense spherical nanoparticles have been found. Suspensions of microparticles after their quick (5 min) heating at 94°C have been examined. The partial melting of smooth microspheres (without thorns) has been established: they lose a part of the DNA so that the details of their structure (ultrastructure) can be discerned. Simultaneously, clusters of nanowires appear after the heating. The molecular mechanisms of the formation of nanoand microparticles are discussed. [ABSTRACT FROM AUTHOR]