Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets.
Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.
Anderson
Nari
N
Murdoch University, South Street, Murdoch 6150, Perth Australia.
Szemes
Marianna
M
O'Brien
Phil
P
de Weerdt
Marjanne
M
Schoen
Cor
C
Boender
Piet
P
Bonants
Peter
P
D004271:DNA, Fungal / Q000235:genetics; D021901:DNA, Intergenic / Q000235:genetics; D007700:Kinetics; D016533:Mycological Typing Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D010838:Phytophthora / Q000145:classification* / Q000235:genetics; D013045:Species Specificity; D044366:Transition Temperature
D004271:DNA, Fungal; D021901:DNA, Intergenic; D015345:Oligonucleotide Probes