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Nucleic Acids Research, 1992, Vol. 20, No. 24 6657-6665
© 1992

CHEMISTRY

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera

Achim E. Karger*, Robert Weiss and Raymond F. Gesteland1

Department of Human Genetics, University of Utah Salt Lake City, UT 84112 USA 1Howard Hughes Medical Institute, University of Utah Salt Lake City, UT 84112 USA

*To whom correspondence should be addressed

Received August 20, 1992. Revised November 19, 1992. Accepted November 19, 1992.

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 blotinylated 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.


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