CHAPTER SIX Nucleic acid hybridization: principles and applications

1. CHAPTER SIXNucleic acid hybridization: principles and applications 생물정보학협동과정 강민호

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.

4. Types of probes

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 Fluorescence microscopy Common Fluorophores

14. Structure of digoxigenin-modified and biotin-modified nucleotides

15. Indirect nonisotopic labeling

16. Nucleic acid hybridization- formation of heteroduplexes

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

18. 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

19. 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

20. The identification of specific sequences in a complex mixture.

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

22. 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

23. Dot blot or slot blot

24. Principles of Southern blot

25. Northern Blot

26. Colony blot hybridization

27. In situ hybridization • Chromosome in situ hybridization • Metaphase or protometaphase chromosomes are probed with labeled DNA . The DNA can be labeled with a fluorochrome (FISH). • Tissue in situ hybridization • Sliced or whole mounted preparations can be probed with RNA probes to detect mRNA expression

28. Tissue In situ hybridization

29. Gridded clone hybridization

30. Construction of DNA and oligo microarrays

31. Gene expression profiling by hybridization

32. Summary I • Hybridization is due to complementarity of DNA strands. • DNA can be labeled various ways • Isotopic and non isotopic • Hybridization can detect identical or similar sequences.

33. Summary II • A variety of techniques utilize hybridization of DNA or RNA probes • ASO • Southern Blot, RFLP, VNTRs, Mutation detection, deletion detection • Northern Blot, tissue specific expression • In situ hybridization • Chromosome location and integrity • Tissue specific expression

34. Summary III • Colony hybridization can be used to identify specific clones. Once you have one clone you can find others that hybridize to it. • Screening of gridded clones . One can identify genomic clones homologous to a cDNA or identify cDNA expressed in a cell line. • Microarrays can be used in many ways to analyze gene expression in various cell types, in response to various stimuli.