Brow M A, Oldenburg M C, Lyamichev V, Heisler L M, Lyamicheva N, Hall J G, Eagan N J, Olive D M, Smith L M, Fors L, Dahlberg J E
Third Wave Technologies Inc., University of Wisconsin, Madison, USA.
J Clin Microbiol. 1996 Dec;34(12):3129-37. doi: 10.1128/jcm.34.12.3129-3137.1996.
We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.
我们在此描述了一种使用结构特异性核酸内切酶Cleavase I分析核酸序列的新方法。我们已将该技术应用于检测和定位与结核分枝杆菌异烟肼耐药相关的突变，以及区分细菌属、种和菌株。此处描述的技术基于这样的观察结果：DNA单链可以呈现特定的构象，这些构象可被诸如Cleavase I之类的结构特异性核酸内切酶检测和切割。产生的片段模式是负责该结构的序列所特有的，因此每个DNA都有其自身的结构指纹。从耐异烟肼和敏感的结核分枝杆菌的katG基因的620 bp片段、大肠杆菌O157:H7、鼠伤寒沙门氏菌、肠炎沙门氏菌、亚利桑那沙门氏菌、宋内志贺氏菌、痢疾志贺氏菌、空肠弯曲菌、人葡萄球菌、沃氏葡萄球菌和金黄色葡萄球菌的16S rRNA基因的5' 350 bp以及包含鼠伤寒沙门氏菌、肠炎沙门氏菌、亚利桑那沙门氏菌、宋内志贺氏菌和痢疾志贺氏菌血清型1、2和8的16S和23S rRNA基因之间基因间区域的约550 bp DNA片段中生成了含有单个5'-荧光素或5'-四甲基罗丹明标记的扩增子。耐异烟肼结核分枝杆菌分离株的katG基因衍生的DNA片段的结构指纹变化被清晰识别，并且可以相对于标记末端映射到实际突变位点。从16S基因产生的清晰模式能够将细菌区分到属的水平，在某些情况下还能区分到种的水平。对沙门氏菌和志贺氏菌属的基因间区域进行Cleavase I分析可区分属、种和血清型。用Cleavase I消化进行结构指纹分析是一种快速、简单且灵敏的核酸序列分析方法，可能在微生物分析中具有广泛的用途。
