Sanjuán Rafael, Daròs José-Antonio
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), C/ Ingeniero Fausto Elio s/n, 46022 València, Spain.
J Virol Methods. 2007 Oct;145(1):71-5. doi: 10.1016/j.jviromet.2007.05.027. Epub 2007 Jun 29.
The study of viroids (plant pathogens constituted by small non-coding RNA) has greatly benefited from the use of site-directed mutagenesis tools. However, compared to viral systems, this technique is complicated by the fact that, usually, infectious cDNAs carry two copies of the viroid genome. A simple method for a one-step site-directed mutagenesis of viroids is described and tested by estimating the rate of mutation incorporation of three random nucleotide substitutions in each Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd). The protocol is essentially based on the original QuickChange Stratagene methodology; dimeric cDNA templates are amplified directly using Pfu DNA polymerase and self-complementary mutagenic primers. The reaction typically yields dimeric, but also monomeric clones which can be easily distinguished by electrophoretic analysis. The data show that approximately 50% of the dimeric clones carry the desired mutation in both viroid copies. Since the proposed protocol is simple technically and rapid compared to previous methods, it could be applied routinely for site-directed mutagenesis studies in viroids.
对类病毒（由小的非编码RNA构成的植物病原体）的研究极大地受益于定点诱变工具的使用。然而，与病毒系统相比，该技术因以下事实而变得复杂：通常，感染性cDNA携带类病毒基因组的两个拷贝。本文描述了一种用于类病毒一步定点诱变的简单方法，并通过估计在每种菊花矮化类病毒（CSVd）和菊花褪绿斑驳类病毒（CChMVd）中三个随机核苷酸替换的突变掺入率进行了测试。该方案基本上基于原始的QuickChange Stratagene方法；使用Pfu DNA聚合酶和自互补诱变引物直接扩增二聚体cDNA模板。该反应通常产生二聚体，但也产生单体克隆，可通过电泳分析轻松区分。数据表明，大约50%的二聚体克隆在两个类病毒拷贝中都携带所需的突变。由于所提出的方案在技术上简单且与以前的方法相比快速，因此它可常规应用于类病毒的定点诱变研究。
