MolBio

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,
to hybridize to each other)

3. - 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.
Standard nucleic acid
hybridization assays

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

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

23. 06_10.jpg

24. Filter hybridization
techniques
Filter hybridization methods
Bacteriophage blotting
Benton-Davis
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