A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I.
A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I.
复制标题
基于 Tn3 转座子诱变的定向 DNA 测序策略：应用于酿酒酵母 I 号染色体上的 ADE1 基因座。
DOI：
10.1093/nar/19.20.5731
复制
发表时间：
1991
影响因子：
14.9
通讯作者：
Hutchison3rd,CA
中科院分区：
文献类型：
--
作者：
Davies,CJ;Hutchison3rd,CA
我们开发了基于大肠杆菌转座子 Tn3 的定向 DNA 测序策略。该转座子几乎没有表现出转座的序列特异性，因此非常适合这项任务。带有噬菌体 f 1 单链复制起点的微型 Tn3 转座子和测序载体均已构建。靶序列诱变后，会产生一个群体，其中每个克隆都有两个 f1 复制起点，其中一个复制起点的位置取决于转座子插入位点。当辅助噬菌体添加到诱变群体中时，每个克隆上存在的两个 f1 起点都会被切割，将包装的链分成两段，每段都包装到单独的噬菌体颗粒中。其中一个片段不包含抗性标记并且丢失，而另一个片段作为具有单个嵌合 f1 起源的删除克隆被恢复。产生一组全向、可变删除的测序克隆，并使用与转座子末端互补的引物对适当大小的克隆进行测序。除了便宜之外，该方法不需要许多体外酶促方法所需的相同程度的技术专业知识。该策略已用于确定酿酒酵母 ADE 1 基因座中 2.6 KB 的核苷酸序列。
We have developed a directed DNA sequencing strategy based upon theEscherichia colitransposon Tn3. This transposon displays little sequence specificity for transposition and is thus well suited to this task. Both mini-Tn3 transposons and sequencing vectors bearing the phage f 1 single stranded origin of replication have been constructed. Upon mutagenesis of a target sequence, a population is produced in which each clone has two f1 origins of replication, one of which is at a variable position depending upon the tranposon insertion site. When helper phage is added to the mutagenised population, the two f1 origins present on each clone are nicked, dividing the packaged strand into two segments, each of which is packaged into a separate phage particle. One of these segments contains no resistance markers and is lost, whilst the other is recovered as a deleted clone with a single chimeric f1 origin. A enidirectionally, variablydeleted set of sequencing clones is produced, and appropriately sized clones are sequenced using a primer complimentary to the transposon end. In addition to being inexpensive, the method does not require the same degree of technical expertise neededfor manyin vitro, enzymatically based methods. The strategy has been used to determine 2.6 kilobases of nucleotide sequence in theSaccharomyces cerevisiae ADE 1locus.