title: 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.
authors.affiliation: a Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry , Russian Academy of Science , ul. Miklukho-Maklaya 16/10, Moscow 117997 , Russia.
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Danilevich
authors.affiliation: b Skryabin Institute of Biochemistry and Physiology of Microorganisms , Russian Academy of Sciences , Pr. Nauki 5, 142290 Pushchino , Moscow Region, Russia.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Machulin
authors.affiliation: a Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry , Russian Academy of Science , ul. Miklukho-Maklaya 16/10, Moscow 117997 , Russia.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Lipkin
authors.affiliation: b Skryabin Institute of Biochemistry and Physiology of Microorganisms , Russian Academy of Sciences , Pr. Nauki 5, 142290 Pushchino , Moscow Region, Russia.
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Kulakovskaya
authors.affiliation: c Beckman Research Institute and Division of Urology and Urologic Oncology , City of Hope National Medical Center , 1500 E. Duarte Rd., Duarte , CA 91010 , USA.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Smith
authors.affiliation: d Winogradsky Institute of Microbiology , Russian Academy of Science , Pr. 60-letiya Oktyabrya 7/2, Moscow 117312 , Russia.
authors.forename: Andrey L
authors.initials: AL
authors.lastname: Mulyukin
mesh_terms: D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry; D017616:Magnesium Compounds / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000379:methods; D012964:Sodium / Q000737:chemistry
chemical_list: D017931:DNA Primers; D011756:Diphosphates; D017616:Magnesium Compounds; D004247:DNA; D012964:Sodium; C034732:magnesium pyrophosphate; D008274:Magnesium
keywords: PCR-generated DNA particles; magnesium pyrophosphate microparticles; polymerase chain reaction; scanning electron microscopy; thermal cycling