Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology

Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase cha...

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Bibliographic Details
Published in:Analytical biochemistry 2005-07, Vol.342 (1), p.93-102
Main Authors: Koch, C.A., Li, P.C.H., Utkhede, R.S.
Format: Article
Language:English
Subjects:
Agarose substrate
Ascomycota - genetics
Ascomycota - isolation & purification
Botrytis - genetics
Botrytis - isolation & purification
Botrytis cinerea
Didymella bryoniae
DNA, Fungal - chemistry
Glass
Microarray
Nucleic Acid Hybridization - methods
Oligonucleotide Array Sequence Analysis - instrumentation
Oligonucleotide Array Sequence Analysis - methods
Plant Diseases - microbiology
Polymerase Chain Reaction
Sepharose - chemistry
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Summary:Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase chain reaction (PCR)-amplified fluorescently labeled DNA extracted from pure culture and from diseased plant tissue. The probes easily distinguished these pathogens from each other without cross reaction. Thickness of the agarose layer and length of the sample DNA were important factors affecting hybridization efficiency of immobilized probe to PCR product. These factors did not affect hybridization with short complementary oligonucleotide. Probes fixed on agarose-coated slides could differentiate samples as readily as probes on nylon but with potentially higher spot density and gave much better signal than probes on silylated slides. The use of plain glass slides, agarose, and a manual arrayer makes this technique useful for developing specialized and inexpensive DNA microarrays on a solid rigid substrate.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2005.04.010