Zhong Shaobin, Dean Antony M
Biotechnology Institute, University of Minnesota, 1479 Gortner Ave, St, Paul, MN 55108, USA.
BMC Microbiol. 2004 Jul 8;4:26. doi: 10.1186/1471-2180-4-26.
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.
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.
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.
插入序列（IS）是能够在原核基因组内及之间进行转座的小DNA片段，常导致插入突变和染色体重排。尽管有几种方法可用于在微生物基因组中定位IS，但它们要么劳动强度大，要么效率低下。在此，我们使用载体PCR来鉴定和绘制在大肠杆菌菌株CGSC6300中发现的八个插入序列（IS1、2、3、4、5、30、150和186）的基因组位置，CGSC6300是MG1655的近亲，其基因组已被测序。
用四碱基切割酶Rsa I消化CGSC6300菌株的基因组DNA，并将所得的限制性片段连接到载体单元上。使用从每个IS的末端向外定向的IS特异性引物和一个载体引物，从MG1655基因组序列中鉴定出的37个IS元件中除一个之外的所有元件扩增侧翼DNA片段。PCR产物的纯化和测序证实它们是与IS相关的侧翼DNA片段，对应于MG1655基因组中已知的IS位置。在CGSC6300菌株中发现了另外七个插入，表明同一实验室菌株（K12分离株）的非常密切相关的分离株在其IS组成上可能不同。另外两个大肠杆菌K12衍生物TD2和TD10也通过载体PCR进行了分析。它们分别共享36个MG1655的IS位点，以及分别有16个和18个额外的插入。
本研究表明，载体PCR是一种快速、高效、可靠的对微生物菌株进行分型以及鉴定和绘制微生物基因组中IS插入位点的方法。与Southern杂交和反向PCR不同，我们的方法仅涉及一次基因组消化和一次连接步骤。然后使用载体PCR同时扩增给定类型的所有IS元件，使其成为一种快速且灵敏的检测基因组中IS元件的手段。快速鉴定微生物基因组的IS组成的能力应有助于在疾病爆发期间对密切相关的病原体进行亚型分析。
