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

Abstract

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.

Polyphosphatase PPN1 of <i>Saccharomyces cerevisiae</i> Is a Deoxyadenosine Triphosphate Phosphohydrolase

“…The preliminary data on the hydrolysis of dNTP mixture suggest the ability of hydrolysis of other dNTP. It should be noted that the PPX1 exopolyphosphatase of S. cerevisiae hydrolyzes neither dATP (this study) nor dNTP mixture [19]. The catabolism of dNTPs is performed by many enzymes.…”

“…No other substrates of this enzyme are known; the pyrophosphatase activity of PPN1 was extremely low [17]. We have attempted to use the pure recombinant PPN1 [18] to reveal PolyP in the PCR reaction products with the high level of pyrophosphate [19]. In these experiments, the treatment of dNTP mixture with PPN1 resulted in Pi release; i.e., PPN1 catalyzed the reaction:…”

Sustained Release of Minor-Groove-Binding Antibiotic Netropsin from Calcium-Coated Groove-Rich DNA Particles

“…Using the circular DNA template, Ф29 DNA polymerase polymerized the elongated strand of DNA. Pyrophosphate (PPi), which is produced during the polymerization process, and Mg 2+ , a cofactor of Ф29 DNA polymerase, were reacted to form magnesium pyrophosphate (Mg 2 PPi), inducing DNAp synthesis with a spherical particle shape (Figure S1) [49,50].…”