Characterization of dye-induced mobility shifts affecting DNA sequencing in poly(ethylene oxide) sieving matrix.
The influence of three classes of fluorescence labels including dipyrrometheneboron difluoride (BODIPY), energy transfer (ET) and conventional fluorescein and rhodamine (ABI), on DNA sequencing has been examined with laser-induced fluorescence detection and poly(ethylene oxide)-filled capillary electrophoresis. DNA sequencing fragments were generated by dye-labeled primer cycle-sequencing reactions in a hot-air thermal cycler. A parameter, relative-induced shift, was introduced to quantify the uniformity of electrophoretic mobilities of these fragments. BODIPY was found to have the smallest, but nonzero, effect for dye-induced nonuniformity. Although ET dyes provided the highest sensitivity due to their unique spectroscopic properties, they were found to lack photostability compared to BODIPY and ABI dyes. Characterization also brings out some important tips for selecting the suitable dye set for the two-channel ratio-based DNA base-calling method.
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Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, 50011, USA.
Yeung
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D053938:DNA Fragmentation; D004586:Electrophoresis / Q000379:methods*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D011092:Polyethylene Glycols; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D011092:Polyethylene Glycols