Quantification of sensitive non-isotopic filter hybridizations using the peroxidase catalyzed luminol reaction.
The development of a sensitive, non-isotopic filter hybridization method based on the peroxidase catalyzed luminol reaction is described. High sensitivity was achieved by optimizing the conditions of the hybridization procedure, the immunochemical detection and the peroxidase/luminol reaction. This resulted in the reproducible detection of 10-30 femtogram of target DNA on blots within minutes when a cooled charge coupled device (CCD) camera was used to record the luminescence signal. After optimalization, the system was successfully applied for the qualitative and quantitative analysis of small amounts of DNA in dot-blots as well as in Southern blot analysis.
van Gijlswijk
R P
RP
Department of Cytochemistry and Cytometry, Sylvius Laboratory, Leiden University, The Netherlands.
Raap
A K
AK
Tanke
H J
HJ
D001710:Biotin; D001713:Biphenyl Compounds; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis*; D016264:Dextran Sulfate; D004076:Digoxigenin; D049454:Firefly Luciferin; D006735:Horseradish Peroxidase; D007462:Iodobenzenes; D008163:Luminescent Measurements; D008165:Luminol; D009693:Nucleic Acid Hybridization; D010781:Photography / Q000295:instrumentation; D011092:Polyethylene Glycols; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013844:Thiazoles; D014462:Ultrafiltration
D001713:Biphenyl Compounds; D007462:Iodobenzenes; D013844:Thiazoles; C077548:6-hydroxybenzothiazole; D011092:Polyethylene Glycols; C050181:4-iodophenol; D008165:Luminol; D049454:Firefly Luciferin; D001710:Biotin; D004247:DNA; D016264:Dextran Sulfate; C019046:4-phenylphenol; D006735:Horseradish Peroxidase; D004076:Digoxigenin