Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.
A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.
Höltke
H J
HJ
Boehringer Mannheim GmbH, Department of Molecular Biology, Penzberg, FRG.
Sagner
G
G
Kessler
C
C
Schmitz
G
G
D000199:Actins / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D004076:Digoxigenin; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis*; D010920:Placenta / Q000737:chemistry; D012313:RNA / Q000032:analysis; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis; D011973:Receptors, LDL / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics
D000199:Actins; D015342:DNA Probes; D007202:Indicators and Reagents; D009696:Nucleic Acids; D015347:RNA Probes; D012333:RNA, Messenger; D011973:Receptors, LDL; D012313:RNA; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D010959:Tissue Plasminogen Activator; D004076:Digoxigenin