New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals. Issue 3 (3rd March 2016)

• Record Type: Journal Article
• Authors: Danilevich, Vasily N.; Machulin, Andrey V.; Lipkin, Alexey V.; Kulakovskaya, Tatyana V.; Smith, Steven S.; Mulyukin, Andrey L.
• Is Part Of: Journal of biomolecular structure & dynamics. Volume 34:Issue 3(2016)
• Page Start: 625
• Page End: 639
• Publication Date: 2016-03-03
• Subjects: polymerase chain reaction -- PCR-generated DNA particles -- thermal cycling -- magnesium pyrophosphate microparticles -- scanning electron microscopy; Biomolecules -- Periodicals; Molecular structure -- Periodicals; Molecular Biology -- Periodicals; Biomechanics -- Periodicals; 572
• DOI: 10.1080/07391102.2015.1040842
• Languages: English
• ISSNs: 0739-1102

Abstract

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.