Microarray of DNA-protein complexes on poly-3-hydroxybutyrate surface for pathogen detection
Microarray of DNA-protein complexes on poly-3-hydroxybutyrate surface for pathogen detection
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DOI：
10.1007/s00216-008-2574-y
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发表时间：
2009-03-01
影响因子：
4.3
通讯作者：
Lee, Sang Yup
中科院分区：
文献类型：
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作者：
Park, Tae Jung;Yoo, Seung Min;Lee, Sang Yup
利用聚3羟基丁酸酯（poly-3-hydroxybutyrate，PHB）解聚酶的底物结合结构域（substrate-binding domain，SBD）作为活性结合基序，将DNA探针特异性固定在PHB表面。为了证明该方法是否可用于临床病原体的检测，病原体特异性生物素标记的DNA探针通过与SBD融合的核心链霉亲和素（cSA）固定。设计了4种模式病原体的特异性15-mer寡核苷酸探针，并利用通用引物PCR制备了目标DNA。将病原体特异性探针和cSA-SBD融合蛋白的复合物通过显微点样固定在涂覆有PHB的载玻片上。该DNA-蛋白质复合物微阵列能够成功地诊断鲍曼不动杆菌、大肠埃希菌、肺炎克雷伯菌和铜绿假单胞菌。此外，在存在其他微生物的情况下，可以诊断特定的病原体。因此，DNA-蛋白质复合物微阵列平台技术，采用聚羟基丁酸酯和SBD在这里报告可以广泛用于检测DNA-DNA和DNA-生物分子的相互作用，而无需合成或化学修饰的生物分子或固体表面。
A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA-protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA-protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA-DNA and DNA-biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface.