Danilevich Vasily N, Machulin Andrey V, Lipkin Alexey V, Kulakovskaya Tatyana V, Smith Steven S, Mulyukin Andrey L
a Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry , Russian Academy of Science , ul. Miklukho-Maklaya 16/10, Moscow 117997 , Russia.
b Skryabin Institute of Biochemistry and Physiology of Microorganisms , Russian Academy of Sciences , Pr. Nauki 5, 142290 Pushchino , Moscow Region, Russia.
J Biomol Struct Dyn. 2016;34(3):625-39. doi: 10.1080/07391102.2015.1040842. Epub 2015 May 20.
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.
这项工作旨在研究聚合酶链式反应（PCR）过程中含DNA的微粒和纳米颗粒形成所涉及的分子机制。在使用Taq聚合酶的PCR反应中，焦磷酸和镁离子在生成具有独特且复杂结构的DNA微粒（MPs）过程中均发挥了重要作用。因此，向PCR混合物中添加嗜热栖热菌焦磷酸酶会抑制这一过程，并导致合成的DNA MPs遭到破坏。在标准PCR条件下，对含有焦磷酸钠（Na-PPi）和镁离子（Mg²⁺）的混合物（不含DNA聚合酶和脱氧核苷三磷酸）进行热循环，会产生由焦磷酸镁（Mg-PPi）组成的结晶状椭圆形或透镜状微盘以及三维MPs。扫描电子显微镜（SEM）显示，所产生的Mg-PPi微粒由相交的盘状结构或其片段组成。它们在形态上相似，但比PCR反应中产生的含DNA的MPs更简单。将脱氧核苷三磷酸、引物或双链DNA掺入Mg-焦磷酸颗粒会导致三维微粒的结构多样化。因此，PCR反应中产生的DNA MPs的异常复杂结构是由Mg-焦磷酸在热循环过程中形成超分子颗粒的独特特性所决定的。我们推测，热循环过程中产生的Mg-焦磷酸颗粒充当了扩增子DNA凝聚的支架。
