Detection of isozymes of deoxyribonucleases I and II on 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 sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluorescence 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 in sensitivity compared with the original DAFO method using ethidium bromide (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 1 microL human serum. This new technique has been named SG-DAFO, after its original dried agarose film overlay method using EB (EB-DAFO).
Iida
R
R
Department of Forensic Medicine, Fukui Medical University, Japan.
Yasuda
T
T
Tsubota
E
E
Nakashima
Y
Y
Sawazaki
K
K
Aoyama
M
M
Matsuki
T
T
Kishi
K
K
D052160:Benzothiazoles; D003850:Deoxyribonuclease I / Q000097:blood*; D003959:Diamines; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000097:blood*; D004996:Ethidium; D005456:Fluorescent Dyes; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000032:analysis*; D008832:Microchemistry; D009930:Organic Chemicals; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D007527:Isoenzymes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C019329:deoxyribonuclease II; D004996:Ethidium