Analysis and Characterization of Nucleic Acids and Proteins

1. Molecular Diagnostics Molecular Diagnostics Analysis and Characterization of Nucleic Acids and Proteins 1

2. 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.

3. Molecular Diagnostics Type Origins Characteristic of starting material Labeling

4. 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.

5. Molecular Diagnostics Random primed labeling with klenow

6. Molecular Diagnostics Nick translation with DNA polymerase I and DNAse I

7. Molecular Diagnostics 3’fill-end labeling by klenow

8. Molecular Diagnostics 5’end labeling with polynucleotide kinase

9. Molecular Diagnostics Run-off transcription from cloned DNA inserts in specialized plasmid vectors

10. 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.

11. Molecular Diagnostics

12. Molecular Diagnostics

13. 13 Molecular Diagnostics Molecular Diagnostics Optimal Hybridization Times General Hybridization Times/ Temperatures ON=overnight

14. 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. 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. 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. 17 Molecular Diagnostics Molecular Diagnostics

18. 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. 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

20. 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. 21 Molecular Diagnostics Molecular Diagnostics Detection Methods • Isotopic labels (3H, 32P, 35S, 125I) • Photographic exposure (X-ray film) • Quantification (scintillation counting, densitometry)

22. 22 Molecular Diagnostics Molecular Diagnostics Detection Methods • Non-isotopic labels (enzymes, lumiphores) • Enzymatic reactions (peroxidase, alkaline phosphatase) • Luminescence (Adamantyl Phosphate derivatives, “Lumi-Phos”)

23. 23 Molecular Diagnostics Molecular Diagnostics Radioactive or chemiluminescent detection (autoradiography film) Chromogenic detection (nitrocellulose membrane) Southern Blot Results

24. 24 Molecular Diagnostics Molecular Diagnostics

25. 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)

26. Molecular Diagnostics Line probe assay (LIPA): • Based on reverse hybridization • Available commercially • Homework: • List at least 3 specific applications for LIPA