Quick Review Cards for Medical Laboratory Science

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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