Microarray of DNA-protein complexes on poly-3-hydroxybutyrate surface for pathogen detection.
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.
Park
Tae Jung
TJ
Department of Chemical and Biomolecular Engineering (BK21 program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
Yoo
Seung Min
SM
Keum
Ki Chang
KC
Lee
Sang Yup
SY
D040981:Acinetobacter baumannii / Q000235:genetics / Q000302:isolation & purification*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D006885:Hydroxybutyrates / Q000737:chemistry*; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D011091:Polyesters / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013499:Surface Properties; D000094122:Polyhydroxybutyrates
D002265:Carboxylic Ester Hydrolases; D015342:DNA Probes; D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D006885:Hydroxybutyrates; D011091:Polyesters; D011993:Recombinant Fusion Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase