An inverse PCR technique to rapidly isolate the flanking DNA of dictyostelium insertion mutants
An inverse PCR technique to rapidly isolate the flanking DNA of dictyostelium insertion mutants
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DOI：
10.1385/mb:26:3:221
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发表时间：
2004-03-01
影响因子：
2.6
通讯作者：
Harwood, AJ
中科院分区：
文献类型：
--
作者：
Keim, M;Williams, RSB;Harwood, AJ
限制性内切酶介导的整合是盘基网柄藻插入突变的一种常用而有效的方法。在该方法中，使用质粒拯救来克隆插入位点侧翼的基因组脱氧核糖核酸（DNA）序列。为了使其有效，必须首先在基因组DNA内找到方便的限制性内切酶位点。这是一个耗时的过程，需要对突变体DNA进行Southern印迹分析。此外，质粒拯救需要转化到高度感受态的大肠杆菌中。由于不稳定的基因组序列、质粒DNA的损伤和外源质粒污染，可能会出现问题。我们已经建立了一个简单而快速的聚合酶链反应为基础的技术，适用于所有的突变体，并规避了Southern印迹分析和质粒拯救的需要。
Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.