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Molecular Diagnostics. Analysis and Characterization of Nucleic Acids and Proteins. 1. PROBES. Single-stranded DNA or RNA molecules used to detect complementary sequences. carry radioactive or chemical markers to facilitate their detection.
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Molecular Diagnostics Molecular Diagnostics Analysis and Characterization of Nucleic Acids and Proteins 1
Molecular Diagnostics PROBES • 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.
Molecular Diagnostics Type Origins Characteristic of starting material Labeling
Molecular Diagnostics DNA LABELING • Incorporation of labeled nucleotides: • Random primed labeling with klenow. • Nick translation with DNA polymerase I and DNAse I. • End labeleing: • 3’end labeling with terminal transferase. • 5’end labeling with polynucleotide kinase.
Molecular Diagnostics Random primed labeling with klenow
Molecular Diagnostics Nick translation with DNA polymerase I and DNAse I
Molecular Diagnostics 3’fill-end labeling by klenow
Molecular Diagnostics 5’end labeling with polynucleotide kinase
Molecular Diagnostics Run-off transcription from cloned DNA inserts in specialized plasmid vectors
Molecular Diagnostics labeling • Isotopic labeling: • often 32P, 33P, 35S or 3H • None isotopic labeling: • Direct nonisotopic labeling: • Often involve incorporation of modified nucleotides containing a fluorophore. • Indirect nonisotopic labeling. • The biotin-streptavidine system • Digoxigenin system.
13 Molecular Diagnostics Molecular Diagnostics Optimal Hybridization Times General Hybridization Times/ Temperatures ON=overnight
14 Molecular Diagnostics Molecular Diagnostics Hybridization Conditions • Three steps of hybridization reaction • Prehybridization to block non-specific binding • Hybridization under appropriate conditions • Post-hybridization to remove unbound probe
15 Molecular Diagnostics Molecular Diagnostics Hybridization Stringency • Stringency is the combination of conditions in which the target is exposed to the probe. • Conditions of high stringency are more demanding of probe/target complementarity. • If conditions of stringency are set too high, the probe will not bind to its target. • Low stringency conditions are more forgiving. • If conditions are set too low, the probe will bind unrelated targets, complicating interpretation of the final results.
16 Molecular Diagnostics Molecular Diagnostics Hybridization Stringency • 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
17 Molecular Diagnostics Molecular Diagnostics
Molecular Diagnostics Several factors affect stringency • Temperature of hybridization • Calculate Tm of the probe sequence • 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. • 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.
19 Molecular Diagnostics Molecular Diagnostics Several factors affect stringency • 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
Molecular Diagnostics • 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.
21 Molecular Diagnostics Molecular Diagnostics Detection Methods • Isotopic labels (3H, 32P, 35S, 125I) • Photographic exposure (X-ray film) • Quantification (scintillation counting, densitometry)
22 Molecular Diagnostics Molecular Diagnostics Detection Methods • Non-isotopic labels (enzymes, lumiphores) • Enzymatic reactions (peroxidase, alkaline phosphatase) • Luminescence (Adamantyl Phosphate derivatives, “Lumi-Phos”)
23 Molecular Diagnostics Molecular Diagnostics Radioactive or chemiluminescent detection (autoradiography film) Chromogenic detection (nitrocellulose membrane) Southern Blot Results
24 Molecular Diagnostics Molecular Diagnostics
25 Molecular Diagnostics Molecular Diagnostics Southern Blot Applications • Genetics, oncology (translocations, gene rearrangements) • Typing/classification of organisms • Cloning/verification of cloned DNA • Forensic, parentage testing (RFLP, VNTR)
Molecular Diagnostics Line probe assay (LIPA): • Based on reverse hybridization • Available commercially • Homework: • List at least 3 specific applications for LIPA