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 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.
Koch
C A
CA
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6.
Li
P C H
PC
Utkhede
R S
RS
D001203:Ascomycota / Q000235:genetics / Q000302:isolation & purification*; D020171:Botrytis / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000737:chemistry; D005898:Glass; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012685:Sepharose / Q000737:chemistry*
D004271:DNA, Fungal; D012685:Sepharose