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- 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 labelednucleic 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
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- 11. Characteristics of radioisotopescommonly 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 anddetection • 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)
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- 16. Nucleic acid hybridization- formationof heteroduplexes
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- 18. Denaturation of DNAresults in an increase of optical density
- 19. Melting curve ofa specific DNA sequence
- 20. Factors affecting Tmof 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 • Ionicstrenght • 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 Lowsalt 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
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- 24. Filter hybridization techniques Filter hybridizationmethods 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
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- 30. Typical hybridization solution •High salt solution (SSC or SSPE) • Blocking agent (Denhardts, salmon sperm DNA, yeast tRNA) • SDS
- 31. Southern Applications • Detectionof 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
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- 35. Colony blot hybridization-example