Use of a capillary electrophoresis instrument with laser-induced fluorescence detection for DNA quantitation. Comparison of YO-PRO-1 and PicoGreen assays.
Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R2 > 0.999) and reproducible (RSD < 9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20 pg and 60 pg, respectively) than the YO-PRO-1 assay (60 pg and 260 pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80 min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required).
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Christelle
C
Guillo
Jerome P
JP
Ferrance
James P
JP
Landers
D001583:Benzoxazoles / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000097:blood; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D006801:Humans; D007834:Lasers; D009930:Organic Chemicals / Q000737:chemistry; D011806:Quinolinium Compounds / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001583:Benzoxazoles; D009930:Organic Chemicals; C099476:PicoGreen; D011806:Quinolinium Compounds; C089813:YO-PRO 1; D004247:DNA