title: Improved DNA microarray detection sensitivity through immobilization of preformed in solution streptavidin/biotinylated oligonucleotide conjugates.
abstract: A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40 min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection.
authors: Immunoassay/Immunosensors Lab, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, NCSR "Demokritos", GR-15310 Aghia Paraskevi, Greece.
authors: E
authors: E
authors: Mavrogiannopoulou
authors: Immunoassay/Immunosensors Lab, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, NCSR "Demokritos", GR-15310 Aghia Paraskevi, Greece.
authors: P S
authors: PS
authors: Petrou
authors: Immunoassay/Immunosensors Lab, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, NCSR "Demokritos", GR-15310 Aghia Paraskevi, Greece.
authors: G
authors: G
authors: Koukouvinos
authors: Molecular Diagnostics Lab, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, NCSR "Demokritos", GR-15310 Aghia Paraskevi, Greece.
authors: D
authors: D
authors: Yannoukakos
authors: Biochemistry Lab, Department of Chemistry, University of Athens, GR-15771 Panepistimiopolis, Athens, Greece.
authors: A
authors: A
authors: Siafaka-Kapadai
authors: M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Kraków, Poland.
authors: K
authors: K
authors: Fornal
authors: M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Kraków, Poland.
authors: K
authors: K
authors: Awsiuk
authors: M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Kraków, Poland.
authors: A
authors: A
authors: Budkowski
authors: Immunoassay/Immunosensors Lab, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, NCSR "Demokritos", GR-15310 Aghia Paraskevi, Greece. Electronic address: skakab@rrp.demokritos.gr.
authors: S E
authors: SE
authors: Kakabakos
mesh_terms: D000818:Animals; D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics; D020029:Base Pairing; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D002417:Cattle; D004252:DNA Mutational Analysis / Q000191:economics / Q000592:standards*; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000295:instrumentation / Q000592:standards*; D009841:Oligonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000592:standards; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry*; D013997:Time Factors
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D009841:Oligonucleotides; D012710:Serum Albumin, Bovine; D001710:Biotin; D019809:Streptavidin
keywords: BRCA1 gene mutations; Biotinylated oligonucleotides; Microarrays; Oligonucleotide immobilization; Streptavidin