Transcript Molecular Diagnostics 21

Molecular Diagnostics
Analysis and Characterization of
Nucleic Acids and Proteins
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Molecular Diagnostics
PROBES
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Single-stranded DNA or RNA molecules used to detect
complementary sequences.
carry radioactive or chemical markers to facilitate their
detection.
can be anywhere from fifteen to thousands of
nucleotides long.
Hybridization reactions using DNA/RNA probes are so
sensitive and selective that they can detect
complementary sequences present at a concentration
as low as one molecule per cell.
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Type
Origins
Characteristic
of starting
material
Labeling
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DNA LABELING
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Incorporation of labeled nucleotides:
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Random primed labeling with klenow.
Nick translation with DNA polymerase I and DNAse I.
End labeleing:
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3’end labeling with terminal transferase.
5’end labeling with polynucleotide kinase.
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Random primed labeling with klenow
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Nick translation with DNA polymerase I
and DNAse I
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3’fill-end labeling by klenow
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5’end labeling with polynucleotide
kinase
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Run-off transcription from cloned DNA inserts
in specialized plasmid vectors
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labeling
Isotopic labeling:
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often 32P, 33P, 35S or 3H
None isotopic labeling:
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A.
Direct nonisotopic labeling:
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B.
Often involve incorporation of modified nucleotides
containing a fluorophore.
Indirect nonisotopic labeling.
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The biotin-streptavidine system
Digoxigenin system.
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General Hybridization Times/
Temperatures
Optimal Hybridization Times
ON=overnight
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Hybridization Conditions
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Three steps of hybridization reaction
 Prehybridization to block non-specific binding
 Hybridization under appropriate conditions
 Post-hybridization to remove unbound probe
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Hybridization Stringency
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Stringency is the combination of conditions in which
the target is exposed to the probe.
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Conditions of high stringency are more demanding of
probe/target complementarity.
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If conditions of stringency are set too high, the probe will not
bind to its target.
Low stringency conditions are more forgiving.
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If conditions are set too low, the probe will bind unrelated
targets, complicating interpretation of the final results.
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Molecular Diagnostics
Hybridization Stringency
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Closely related genes are not identical in
sequence, but are similar
Conserved sequence relationship is indicator of
functional importance
Use lower temperature hybridization to identify
DNAs with limited sequence homology: reduced
stringency
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Several factors affect stringency
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Temperature of hybridization
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Calculate Tm of the probe sequence
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The hybridization temperature of oligonucleotide probes is about
5C below the melting temperature
Salt concentration of the hybridization buffer
The concentration of denaturant such as formamide
in the buffer.
The nature of the probe sequence can also impinge
on the level of stringency.
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A probe with a higher percentage of G and C bases will
bind under more stringent conditions than one with greater
numbers of A and T bases.
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Molecular Diagnostics
Several factors affect stringency
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Amount of genomic DNA
Proportion of the genome that is complementary
to the probe
Size of the probe (short probe = low signal)
Labeling efficiency of the probe
Amount of DNA transferred to membrane
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High Stringency for well matched hybrids
 High temp (65o-68oC) or 42oC in presence of 50%
formamide
 Washing with low salt (0.1X SSC), high temp (25oC)
Low Stringency
 Low temp, low formamide
 Washing with high salt
Formamide concentration increases stringency.
Low salt increases stringency.
Heat increases stringency.
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Detection Methods
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Isotopic labels (3H, 32P, 35S,
125I)
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Photographic exposure (X-ray film)
Quantification (scintillation
counting, densitometry)
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Detection Methods
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Non-isotopic labels (enzymes,
lumiphores)
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Enzymatic reactions (peroxidase,
alkaline phosphatase)
Luminescence (Adamantyl
Phosphate derivatives, “Lumi-Phos”)
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Southern Blot Results
Radioactive or
chemiluminescent detection
(autoradiography film)
Chromogenic detection
(nitrocellulose membrane)
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Southern Blot Applications
Genetics, oncology (translocations, gene
rearrangements)
 Typing/classification of organisms
 Cloning/verification of cloned DNA
 Forensic, parentage testing (RFLP, VNTR)
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Line probe assay (LIPA):
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Based on reverse hybridization
Available commercially
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Homework:
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List at least 3 specific applications for
LIPA