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 Mg²⁺ 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 Mg²⁺-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.

Keywords:

• polymerase chain reaction
• PCR-generated DNA particles
• thermal cycling
• magnesium pyrophosphate microparticles
• scanning electron microscopy

Acknowledgments

The authors thank Prof. V.I. Duda for consults and discussion, Dr V.V. Sorokin, and A.S. Shebanova for assistance in electron microscopy studies.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by Russian Foundation for Basic Research [grant number 14-08-00585].