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通过Hirt提取和Southern印迹分析腺相关病毒的复制中间体

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

作者信息

Bardelli Martino, Zarate-Perez Francisco, Agundez Leticia, Jolinon Nelly, Michael Linden R, Escalante Carlos R, Henckaerts Els

机构信息

Department of Infectious Diseases, King's College London, London, United Kingdom.

Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA.

出版信息

Bio Protoc. 2017 May 5;7(9). doi: 10.21769/BioProtoc.2271.

DOI:10.21769/BioProtoc.2271
PMID:28660237
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5484404/
Abstract

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

摘要

腺相关病毒（AAV）是一种小型单链DNA病毒，它需要辅助病毒（如腺病毒或疱疹病毒）的存在才能有效地复制其基因组。AAV DNA通过滚发夹机制进行复制（沃德，2006年），并且在复制过程中可以检测到几种DNA中间体。本详细方案描述了如何使用改良的Hirt提取物（Hirt，1967年）方法和Southern印迹法（Southern，1975年）分析AAV复制过程中形成的AAV DNA中间体。

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本文引用的文献

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7
The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity.腺相关病毒（AAV）的起始结合蛋白Rep68是一种具有DNA解旋酶活性的ATP依赖性位点特异性核酸内切酶。
Cell. 1990 May 4;61(3):447-57. doi: 10.1016/0092-8674(90)90526-k.
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Detection of specific sequences among DNA fragments separated by gel electrophoresis.在通过凝胶电泳分离的DNA片段中检测特定序列。
J Mol Biol. 1975 Nov 5;98(3):503-17. doi: 10.1016/s0022-2836(75)80083-0.
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Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis.正负链交替的串联体是腺病毒相关病毒DNA合成的中间体。
Proc Natl Acad Sci U S A. 1976 Mar;73(3):742-6. doi: 10.1073/pnas.73.3.742.