Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.
Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.
Karger
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AE
Department of Human Genetics, University of Utah, Salt Lake City 84112.
Weiss
R
R
Gesteland
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RF
D000218:Adamantane / Q000031:analogs & derivatives; D000469:Alkaline Phosphatase / Q000235:genetics*; D001345:Autoradiography / Q000379:methods; D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000235:genetics*; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D010781:Photography / Q000379:methods
D007202:Indicators and Reagents; D008567:Membranes, Artificial; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D001710:Biotin; D004247:DNA; D000469:Alkaline Phosphatase; D000218:Adamantane