DNA Hybridization Assays Using Temperature Gradient Focusing and Peptide Nucleic Acids

Two types of DNA hybridization assays are demonstrated with temperature gradient focusing (TGF) and peptide nucleic acids (PNAs). In TGF, the application of a controlled temperature gradient along the length of a microchannel filled with an appropriate temperature-dependent buffer results in the for...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2004-10, Vol.126 (41), p.13474-13479
Main Authors: Balss, Karin M, Ross, David, Begley, Heather C, Olsen, Kimberly G, Tarlov, Michael J
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Buffers
DNA Mutational Analysis - methods
DNA, Single-Stranded - chemistry
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
Microscopy, Fluorescence
Nucleic Acid Hybridization - methods
Nucleic acids
Peptide Nucleic Acids - chemistry
Temperature
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Summary:Two types of DNA hybridization assays are demonstrated with temperature gradient focusing (TGF) and peptide nucleic acids (PNAs). In TGF, the application of a controlled temperature gradient along the length of a microchannel filled with an appropriate temperature-dependent buffer results in the formation of a gradient in both the electric field and electrophoretic velocity. Ionic species move in this gradient and concentrate at a unique point where the total velocity sums to zero. The first assay is a mixing assay in which PNA is allowed to flow through spatially focused DNA targets within a capillary. The second assay detects single base pair mutations (SBPM) by monitoring the fluorescence intensity of PNA/DNA duplexes as a function of temperature within the capillary. The SBPM analysis can be performed in less than 5 min with 100-fold more dilute analyte compared to conventional UV melting measurements.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja030667w