Micro- and nanoparticles of condensed DNA Formed in PCR with Taq polymerase and plasmid DNA as a template
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Formation of micro- and nanoparticles of condensed DNA during PCR with microbial genomic DNA or plasmid DNA as templates was reported previously. Initially, the microparticles were formed using a thermostable KlenTaq polymerase, which is a deletion variant of Taq polymerase. The present work shows that Taq polymerase is also capable of efficient formation of micro- and nanoparticles of condensed DNA in PCR. Electron microscopy revealed a number of morphological types (more than four) of microparticles produced in PCR with different reaction buffers in the presence of Taq polymerase and different plasmid DNAs as a template. In the case of some kinds of amplicons, an increase in the number of thermal cycles was shown to result in production of numerous nanowires and electron-dense spherical nanoparticles. The PCR conditions for preferential formation of discs (or ellipsoids) a few micrometers in diameter and several dozens of nanometers in thickness were determined. The structure of microparticles formed in the presence of Taq polymerase was found to depend on the level of synthesis of single-stranded DNA fragments in PCR. Experiments with nuclease S1 revealed that, along with double-stranded DNAs of the amplicon, micro- and nano-particles contained single-stranded DNA fragments, which were absolutely necessary for their formation. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed.
Keywordspolymerase chain reaction (PCR) Taq polymerase KlenTaq polymerase DNA condensation microparticles and nanoparticles of condensed DNA epifluorescence microscopy electron microscopy
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