Detection of plasmids using DNA and RNA probes and the light-addressable potentiometric sensor

Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using...

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Bibliographic Details
Published in:Journal of biochemical and biophysical methods 1997-12, Vol.35 (3), p.197-202
Main Authors: Dill, Kilian, Chan, Samuel D.H, Gibbs, Thomas W
Format: Article
Language:English
Subjects:
Biotin
DNA Probes
DNA, Viral - analysis
Fluorescein
Hybridization
Light addressable potentiometric sensor (LAPS)
Plasmid detection
Plasmids - chemistry
Potentiometry - methods
RNA Probes
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Summary:Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using the urease-conjugate of an anti-fluorescein antibody via the light-addressable potentiometric sensor. The output of the silicon-chip sensor is in the form of a rate, μV/s, and is directly proportional to the quantity of hybridized DNA captured on the membrane. Use of the larger probes (for β-actin cDNA target) results in detection of intact plasmid at the level of ∼10 6 target molecules; complementary DNA could be detected at similar levels. When the smaller probes are utilized (pGEM or pBSActB targets), plasmid fragmented targets could be detected only at levels 200 times greater than those observed when using the larger RNA probes.
ISSN:0165-022X
1872-857X
DOI:10.1016/S0165-022X(97)00038-9