Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR -: art. no. 26
Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR -: art. no. 26
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DOI：
10.1186/1471-2180-4-26
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发表时间：
2004-07-08
期刊：
影响因子：
4.2
通讯作者：
Dean, AM
中科院分区：
文献类型：
--
作者：
Zhong, SB;Dean, AM
背景：插入序列是能够在原核生物基因组内部和基因组之间转位的小DNA片段，经常引起插入突变和染色体重排。虽然有几种方法可用于定位微生物基因组中的ISs，但它们要么是劳动密集型的，要么是效率低下的。本研究利用载体PCR技术对大肠杆菌CGSC6300中发现的8个插入序列（IS1、2、3、4、5、30、150和186）的基因组位置进行了鉴定和定位。CGSC6300是已测序的MG1655的近亲。结果：菌株CGSC6300的基因组DNA用四碱基切割机Rsa I进行酶切，得到的限制性内切片段连接到载体单元上。利用从每个IS末端向外的IS特异性引物和矢量引物，从MG1655基因组序列中鉴定的37个IS元件中扩增出侧翼DNA片段。PCR产物的纯化和测序证实它们是IS相关的侧翼DNA片段，对应于MG1655基因组中已知的IS位置。在菌株CGSC6300中发现了7个额外的插入，这表明同一实验室菌株（K12分离物）的非常密切相关的分离物可能在IS补体上存在差异。另外两种大肠杆菌K12衍生物TD2和TD10也用载体PCR进行了分析。它们共享36个MG1655 IS位点，并分别有16个和18个额外的插入。结论：载体PCR是一种快速、高效、可靠的微生物分型方法，可用于微生物基因组中is插入位点的鉴定和定位。不像南方杂交和反PCR，我们的方法只涉及一个基因组消化和一个连接步骤。然后使用Vectorette PCR同时扩增给定类型的所有is元件，使其成为快速和敏感的方法来调查基因组中的is元件。快速识别微生物基因组的IS补体的能力应有助于在疾病暴发期间对密切相关的病原体进行分型。
Background: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced.Results: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively.Conclusion: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.