Haddad Yazan, Dostalova Simona, Kudr Jiri, Zitka Ondrej, Heger Zbynek, Adam Vojtech
Department of Chemistry and Biochemistry, Mendel University in Brno; Central European Institute of Technology, Brno University of Technology.
Department of Chemistry and Biochemistry, Mendel University in Brno; Central European Institute of Technology, Brno University of Technology;
J Vis Exp. 2017 Nov 9(129):56815. doi: 10.3791/56815.
Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.
使用磁性颗粒分离DNA在生物技术和分子生物学研究中是一个非常重要的领域。本方案描述了通过动态光散射（DLS）和电泳光散射（ELS）对DNA与磁性颗粒结合的评估。DLS分析提供了关于颗粒物理化学性质的有价值信息，包括粒径、多分散性和zeta电位。后者描述了颗粒的表面电荷，其在诸如DNA等材料的静电结合中起主要作用。在此，进行了一项比较分析，研究了纳米颗粒和微米颗粒的三种化学修饰及其对DNA结合和洗脱的影响。研究了用支化聚乙烯亚胺、原硅酸四乙酯和（3-氨丙基）三乙氧基硅烷进行的化学修饰。由于DNA带负电荷，预计DNA结合后颗粒表面的zeta电位会降低。聚集体的形成也应会影响粒径。为了研究这些颗粒在DNA分离和洗脱中的效率，将颗粒与DNA在低pH（约6）、高离子强度和脱水环境中混合。在磁体上洗涤颗粒，然后用Tris-HCl缓冲液（pH = 8）洗脱DNA。使用定量聚合酶链反应（PCR）估计DNA拷贝数。对zeta电位、粒径、多分散性和定量PCR数据进行评估和比较。DLS是一种有洞察力的辅助分析方法，为筛选用于DNA分离的颗粒的过程增添了新的视角。
