Hybridization Assayscontinued
Molecular Diagnostics
(MDx) Review 562
ASSAY PRINCIPLE APPLICATION
In situ hybridization
DNA chip technology (microarrays)
Cytogenetic studies, e.g., HER2/neu
oncogenes in breast cancer, chromo-
some translocations in leukemia
Gene profiles associated with certain
cancers/diseases, classification of
leukemias, tumor staging, determi-
nation of drug resistance in HIV
Target nucleic acid detected in intact cells. Tissue (fresh,
frozen, or paraffin-embedded) or cells fixed on glass
slide. Probe added. Attaches to complementary DNA
sequences. If fluorescent-labeled probe used, known as
fluorescent in situ hybridization (FISH). Viewed with
fluorescent microscope.
Multiple probes (up to 10^5 –10^6 ) immobilized at specific
locations on solid support (glass slide, silicone chip). After
amplification, sample & control nucleic acids labeled with
2 different fluorescent dyes & loaded onto chip. Sample &
control compete to hybridize to chip based on relative
amounts. Unbound strands of target washed away.
Hybridized nucleic acid detected with fluorescent
detector. Relative colors measured. Thousands of reac-
tions can be performed at same time.
