Hybridization Assays
Molecular Diagnostics
(MDx) Review 561
ASSAY PRINCIPLE APPLICATION
Solid-phase hybridization
Liquid-phase hybridization
Detection of mutations associated
with genetic diseases, e.g., sickle
cell anemia
Infectious diseases, e.g., HCV geno-
typing, Mycobacteriaspeciation
Detection of bacterial rRNA in
clinical specimens or cultures
Dot/slot blot. Sample applied to membrane. Membrane
heated to denature DNA. Labeled probe added. Unhy-
bridized probe washed away. Hybridized probe visualized
by autoradiography or enzyme assay. Pos rxn indicates
presence of target sequence.
Sandwich hybridization. Unlabeled probe bound to mem-
brane or well surface (capture probe). Sample applied. If
target sequence is present, anneals to capture probe. Labeled
probe (signal-generating probe) that anneals to different site
on target added. More specific than dot blot. Can be carried
out in microtiter plates, so adaptable to automation.
Line probe assays (LiPA). Probes attached to strips, amplified
target added. Unbound target washed away. Hybridized
complexes visualized with Biotin-Streptavidin method.
Target nucleic acid & probe interact in aqueous solution.
Rapid & more sensitive than solid phase. Uses smaller
sample size. Adaptable to automation.
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