Szpara Moriah
Department of Biochemistry & Molecular Biology, The Huck Institutes of the Life Sciences, Pennsylvania State University, W-208 Millennium Science Complex (MSC), University Park, PA, 16802, USA,
Methods Mol Biol. 2014;1144:31-41. doi: 10.1007/978-1-4939-0428-0_3.
As an inanimate virus, herpes simplex virus type 1 (HSV-1) necessarily encodes all of its functions in its DNA. Isolation of pure viral DNA allows multiple downstream applications, including the creation of recombinant HSV strains, cloning of selected regions, and sequencing of viral DNA. The term nucleocapsid refers to the combination of the viral genome with the enclosing capsid; these viral genomes are necessarily linear and have been packaged for egress, even if they are not yet released from the cell. In contrast, viral DNA that is not associated with capsids may include episomal or concatenated forms and may have modifications such as histones that are added within cells. During this protocol, the viral capsid protects the HSV genome from reagents that strip away and destroy most cellular contaminants. This procedure describes the isolation of viral nucleocapsids and their subsequent dissolution to purify clean, linear HSV DNA.
作为一种无生命的病毒，单纯疱疹病毒1型（HSV-1）必然在其DNA中编码所有功能。分离纯病毒DNA可实现多种下游应用，包括构建重组HSV毒株、克隆选定区域以及对病毒DNA进行测序。核衣壳一词指的是病毒基因组与包围它的衣壳的组合；这些病毒基因组必然是线性的，并且即使尚未从细胞中释放出来，也已被包装好以便排出。相比之下，未与衣壳结合的病毒DNA可能包括游离形式或串联形式，并且可能有诸如在细胞内添加的组蛋白等修饰。在本实验方案中，病毒衣壳可保护HSV基因组免受去除和破坏大多数细胞污染物的试剂的影响。本方法描述了病毒核衣壳的分离及其随后的溶解，以纯化纯净的线性HSV DNA。
