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1 July 1990 Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera
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Abstract
Fluorescent and chemiluminescent detection of DNA hybrids on polymer membranes has been investigated using a cryogenically cooled, slow readout, two dimensional CCD camera in an imaging mode. The fluorescent background characteristics of commercially available nylon blotting membranes and a polypropylene membrane modified to bind DNA have been studied. The polypropylene membrane exhibits a 15-fold increase in DNA binding, 3-fold less background fluorescence and less background noise than nylon blotting membranes. However the detection limits determined from vacuum slot blots of crosslinked fluoresceinlabelled oligonucleotides show that the improved qualities of the polypropylene support do not result in a lower detection limit. This is mainly due to background noise arising from sources other than the membrane itself during the blotting/washing procedure and to a low signal-to-amount of DNA ratio with the polypropylene membrane. The lowest amount of fluorescein-labelled oligonucleotide detectable is 1.4 femtomol, with a typical exposure time of 10 minutes to image a 6x9 cm area of the membrane. The detection of chemiluminescence was done using a biotin-avidin complex in combination with an enzymatic assay. The assay was carried out after hybridization with biotin-labelled probes on vacuum slot blots with crosslinked target DNA. The detection limit is 0.12 femtomol of DNA target, a result obtained after 30 mm exposure. Further improvements are necessary to image sequencing blots with typically 1 femtomol or less of DNA per band in an acceptable exposure time.
© (1990) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Achim Karger, Jeffrey T. Ives, Robert B. Weiss, Joel M. Harris, and Raymond F. Gesteland "Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera", Proc. SPIE 1206, New Technologies in Cytometry and Molecular Biology, (1 July 1990); https://doi.org/10.1117/12.17814
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