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Proceedings Paper

Imaging of fluorescent and chemiluminescent DNA hybrids using a 2-D CCD camera
Author(s): Achim Karger; Jeffrey T. Ives; Robert B. Weiss; Joel M. Harris; Raymond F. Gesteland
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Paper 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.

Paper Details

Date Published: 1 July 1990
PDF: 12 pages
Proc. SPIE 1206, New Technologies in Cytometry and Molecular Biology, (1 July 1990); doi: 10.1117/12.17814
Show Author Affiliations
Achim Karger, Univ. of Utah (United States)
Jeffrey T. Ives, Howard Hughes Medical Institute (United States)
Robert B. Weiss, Howard Hughes Medical Institute (United States)
Joel M. Harris, Univ. of Utah (United States)
Raymond F. Gesteland, Howard Hughes Medical Institute (United States)

Published in SPIE Proceedings Vol. 1206:
New Technologies in Cytometry and Molecular Biology
Gary C. Salzman, Editor(s)

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