Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots.
A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 x 10(-18) mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images.
Karger
A E
AE
Department of Human Genetics, University of Utah, Salt Lake City.
Weiss
R
R
Gesteland
R F
RF
D000469:Alkaline Phosphatase; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004586:Electrophoresis; D007091:Image Processing, Computer-Assisted; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids; D017422:Sequence Analysis, DNA / Q000379:methods*
D009838:Oligodeoxyribonucleotides; D004247:DNA; D000469:Alkaline Phosphatase