Identification of non-specific hybridization using an empirical equation fitted to non-equilibrium dissociation curves

Non-equilibrium dissociation curves (NEDCs) have the potential to identify non-specific hybridizations on high throughput, diagnostic microarrays. We report a simple method for the identification of non-specific signals by using a new parameter that does not rely on comparison of perfect match and m...

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Bibliographic Details
Published in:Journal of microbiological methods 2012-07, Vol.90 (1), p.29-35
Main Authors: Baushke, Samuel W., Stedtfeld, Robert D., Tourlousse, Dieter M., Ahmad, Farhan, Wick, Lukas M., Gulari, Erdogan, Tiedje, James M., Hashsham, Syed A.
Format: Article
Language:English
Subjects:
Bacteria - chemistry
Bacteria - genetics
Biological and medical sciences
data collection
dissociation
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
equations
fluorescence
Functional gene
Fundamental and applied biological sciences. Psychology
genes
Hybridization
Kinetics
melting point
microarray technology
Microarrays
Microbiology
Non-equilibrium dissociation curve
nucleic acid hybridization
Nucleic Acid Hybridization - methods
ribosomal RNA
RNA, Ribosomal, 16S - chemistry
RNA, Ribosomal, 16S - genetics
sequence homology
Specific dissociation temperature
temperature
Transition Temperature
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Summary:Non-equilibrium dissociation curves (NEDCs) have the potential to identify non-specific hybridizations on high throughput, diagnostic microarrays. We report a simple method for the identification of non-specific signals by using a new parameter that does not rely on comparison of perfect match and mismatch dissociations. The parameter is the ratio of specific dissociation temperature (Td-w) to theoretical melting temperature (Tm) and can be obtained by automated fitting of a four-parameter, sigmoid, empirical equation to the thousands of curves generated in a typical experiment. The curves fit perfect match NEDCs from an initial experiment with an R2 of 0.998±0.006 and root mean square of 108±91 fluorescent units. Receiver operating characteristic curve analysis showed low temperature hybridization signals (20–48°C) to be as effective as area under the curve as primary data filters. Evaluation of three datasets that target 16S rRNA and functional genes with varying degrees of target sequence similarity showed that filtering out hybridizations with Td-w/Tm
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2012.04.003