DETECTION OF ISOZYMES OF DEOXYRIBONUCLEASE-I AND DEOXYRIBONUCLEASE-IION ELECTROPHORESED GELS WITH PICOGRAM SENSITIVITY USING SYBR GREEN I

A highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described, A dried agarose film sh eet containing DNA as a substrate and a buffer reagent was placed in c ontact with the gel surface after electrophoresis (DAFO method, Yasuda ct al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incuba tion period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluore scence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32- to 128-fold increase i n sensitivity compared with the original DAFO method using ethidium br omide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from I mu L human serum. This new tec hnique has been named SG-DAFO, after its original dried agarose film o verlay method using EB (EB-DAFO).