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 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.
Balss
Karin M
KM
National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, USA.
Ross
David
D
Begley
Heather C
HC
Olsen
Kimberly G
KG
Tarlov
Michael J
MJ
D002021:Buffers; D004252:DNA Mutational Analysis / Q000379:methods; D004277:DNA, Single-Stranded / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D013696:Temperature
D002021:Buffers; D004277:DNA, Single-Stranded; D020135:Peptide Nucleic Acids