Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.
Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.
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人工转座子在体外有效整合到质粒靶标中：DNA 作图、测序和遗传分析的有用工具。
DOI：
10.1093/nar/22.18.3765
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发表时间：
1994
影响因子：
14.9
通讯作者：
Boeke,JD
中科院分区：
文献类型：
--
作者：
Devine,SE;Boeke,JD
我们开发了有效的方法来创建人工转座子并将这些转座子在体外插入质粒靶标中，主要用于 DNA 作图和测序的目的。一种新型质粒已被设计用于将几乎任何 DNA 序列或序列组合转化为人工转座子；因此，可以轻松设计和构建包含任何所需特征的定制转座子。然后，利用酵母 Ty1 病毒样颗粒中存在的整合酶活性，将此类转座子在体外有效插入质粒靶标中。单个体外整合反应在反应的复杂性方面类似于简单的限制性消化，在 DNA 靶分子内产生数千个可恢复的插入事件；该频率接近典型质粒中每个磷酸二酯键一个插入。重要的是，转座子插入是从质粒靶标上携带的 DNA 插入的所有区域中恢复的，这表明整合是一个随机或近乎随机的过程。由于其多功能性，该技术提供了一种生成所需结构的重组 DNA 分子的通用方法。我们通过开发定制的人工转座子，将新的引物结合位点插入克隆载体携带的 DNA 插入片段的内部区域，使该系统适用于 DNA 测序。转座子插入已经在质粒上携带的几种不同的酵母和人类 DNA 插入片段中产生，从而可以从这些插入片段中有效地恢复序列信息。我们的结果证明了该方法对于小型和大规模 DNA 测序以及一般 DNA 重组的整体实用性，并表明它可以适用于包括功能遗传分析在内的许多其他应用。
We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targetsin vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets,in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A singlein vitrointegration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.