Nucleic Acid Hybridization

2. Nucleic Acid Hybridization • Nucleic acid hybridization is a fundamental tool in molecular genetics which takes advantage of the ability of individual single-stranded nucleic acid molecules to form double stranded molecules (that is, tohybridize to each other)

3. Standard nucleic acid hybridization assays • A labeled nucleic acid - a probe - to identify • related DNA or RNA molecules • Complex mixture of unlabeled nucleic acid • molecules- the target • Base complementarity with a high degree of • similarity between the probe and the target.

5. Probes • DNA labelling • 5’ • 3’ • Uniform labeling • Nick translation • Random primer • PCR-mediated labeling • RNA labelling • In vitro transcription of a cloned DNA insert • Different probes • Radioactive labeling or isotopic labeling • Nonradioactive labeling or nonisotopic labeling

6. Kinase end-labeling of oligonucleotides

7. Fill-in end labeling

8. Nick translation

9. Random primed labeling

10. Riboprobes

11. Characteristics of radioisotopes commonly used for labeling DNA and RNA probes Radioisotope Half-life Decay-type Energy of emission 3H 12.4 years b- 0.019 MeV 32P 14.3 days b- 1.710 MeV 33P 25.5 days b- 0.248 MeV 35S 87.4 days b- 0.167 MeV

12. Nonisotopic labeling and detection • The use of nonradioactive labels has several advantages: • safety • higher stability of a probe • efficiency of the labeling reaction • detection in situ • less time taken to detect signal • Major types • Direct nonisotopic labeling (ex. nt labeled with a fluorophore) • Indirect nonisotopic labeling (ex. biotin.-streptavidin system)

13. Structure of fluorophores

14. Structure of digoxigenin-modified nucleotides

15. Indirect nonisotopic labeling

16. Nucleic acid hybridization- formation of heteroduplexes

17. Ultraviolet absortion spectrum of DNA

18. Denaturation of DNA results in an increase of optical density

19. Melting curve of a specific DNA sequence

20. Factors affecting Tm of nucleic acid hybrids • Destabilizing agents (ex. formamide, urea) • Ionic strenght • Base composition (G/C%, repetitive DNA) • Mismatched base pairs • Duplex lenght Different equations for calculating Tm for: • DNA-DNA hybrids • DNA-RNA hybrids • RNA-RNA hybrids • Oligonucleotide probes

21. Factors affecting the hybridization for nucleic acids in solution (annealing) • Temperature • Ionic strenght • Destabilizing agents • Mismatched base pairs • Duplex lenght • Viscosity • Probe complexity • Base composition • pH

22. Stringency High temperature Low salt concentration High denaturant concentration Sequence lenght Tm High strigency Perfect match non-complementary sequences Perfect match complementary sequences Sequence G/C content Low strigency Low temperature High salt concentration Low denaturant concentration

23. 06_10.jpg

24. Filter hybridization methods Bacteriophage blotting Benton-Davis Filter hybridization techniques Bacterial colony blotting Grunstein-Hogness Slot/Dot blotting Northern analysis Southern analysis

25. Filters or Membranes • Nitrocellulose • Nylon • Positive charged nylon (hybond) • PVDF (hydrophobic polyvinylidene difloride) • Different properties: • Binding capacity (mg nucleic acids/cm2) • Tensile strenght • Mode of nucleic acid attachment • Lower size limit for efficient nucleic acid retention

26. Principles of Southern blot

27. 06_12_2.jpg

28. Southern Blotting Apparatus

29. Depurination/Denaturation of DNA

30. Typical hybridization solution • High salt solution (SSC or SSPE) • Blocking agent (Denhardts, salmon sperm DNA, yeast tRNA) • SDS

31. Southern Applications • Detection of DNA rearrangements and deletions found in several diseases • Identification of structural genes (related in the same species (paralogs) or in different species (orthologs)) • Construction of restriction maps

32. Southern applications- example

33. Colony blot hybridization-1

34. Colony blot hybridization-2

35. Colony blot hybridization- example