Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments.
Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide.
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Department of Chemistry, Duke University, Durham, NC 27708, USA.
McGown
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D001483:Base Sequence; D002021:Buffers; D002482:Cellulose / Q000031:analogs & derivatives*; D004396:Coloring Agents; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002021:Buffers; D004396:Coloring Agents; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D002482:Cellulose; C002283:hydroxyethylcellulose; D004247:DNA