Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection.
A method has been developed by which enzymatically incorporated fluorophore-labeled nucleotide sites in nucleic acid can be quantitated by degradation of nanogram quantities of DNA followed by capillary gel electrophoretic analysis with fluorescence detection. In this way the differing relative labeling densities achieved using either C5-substituted dUTP's or N4-substituted dCTP's were determined. The method has proven to be very useful in obtaining quantitative analytical data from the small quantities of complex molecules produced in nick translations. Various polymerization conditions using DNA polymerase I were examined to determine optimal labeling density. Simultaneous copolymerization of green fluorescing dCTP and dUTP nucleotides were undertaken in an attempt to maximize labeling density.
Cruickshank
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Vysis Inc., 3100 Woodcreek Drive, Downers Grove, Illinois 60515, USA.
D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000032:analysis* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D019793:Fluorescein; D005456:Fluorescent Dyes; D006801:Humans; D009711:Nucleotides / Q000032:analysis / Q000378:metabolism; D013997:Time Factors
D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D009711:Nucleotides; D004247:DNA; D019793:Fluorescein