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SeaKem® LE Agarose

SeaKem ® LE琼脂糖

Company: Lonza
Catalog#: 50000
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Determination of Mutation Frequency During Viral DNA Replication
[Abstract]  This protocol is a simple method for evaluating mutation frequency during African swine fever virus (ASFV) replication, although it could be used also for other DNA viruses (poxvirus, herpesvirus, mimivirus, etc) with minor modifications. In the original Carrascosa et al. (1982), the protocol was carried out with two cloned viruses, BA71Vc (a purified clone from BA71V wild type strain) and vΔpolX (lacking the reparative polymerase, pol X, gene), and two different cell types that can be infected by ASFV, Vero cells and swine macrophages. To facilitate the sequence comparison, a genome fragment containing the B646L gene was amplified by PCR and blunt-end cloned. This gene codes for the major capsid protein (p72) and multiple sequences can be found in the database, so the ... [摘要]  该协议是用于评估非洲猪瘟病毒(ASFV)复制期间的突变频率的简单方法,尽管其也可以用于其它具有微小修改的DNA病毒(痘病毒,疱疹病毒,mimivirus,等)。 在原始Carrascosa等(1982)中,使用两种克隆的病毒BA71Vc(来自BA71V野生型菌株的纯化克隆)和vΔpolX(缺乏修复聚合酶,pol X, 基因)和可被ASFV,Vero细胞和猪巨噬细胞感染的两种不同细胞类型。 为了便于序列比较,通过PCR扩增含有B646L基因的基因组片段,并平端克隆。 该基因编码主要衣壳蛋白(p72),并且可以在数据库中发现多个序列,因此可以将发现的突变与天然基因变异进行比较。 克隆的片段可以直接从细菌菌落或从小量制备纯化的DNA测序。