A Novel Method for Real-Time, Continuous, Fluorescence-Based Analysis of Anti-DNA Abzyme Activity in Systemic Lupus
Figure 5
Continuous measurement of oligo 18-mer hydrolysis by DNAse I by fluorescence assay. *Analysis of DNAse I positive control via continuous, fluorescence-based assay on Fluoroskan II (Finstruments) demonstrates that 5′-6-FAM-ATATAGCGC5T5-DQ1-3′ 18-mer hydrolysis probe is cleaved by DNAse I and that adequate quenching to allow differentiation between cut and uncut probe was achieved. Oligo blank contained all reaction components except for DNAse I (25 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, pH 7.5, and 1 μg/mL 5′-6-FAM-ATATAGCGC5T5-DQ1-3′ 18-mer hydrolysis probe). Results were consistent across all trials. DNAse I trials 1, 2, and 3 are displayed as representative trials. Pooled fluorescence data showed statistical significant difference from oligo blank (F = 29.78, ).
