1. DNA-based Tools ASO (Allele Specific Oligonucleotides) –Used to detect specific alleles. –Can determine if a person is homozygous or heterozygous for a particular allele

4. DNA-based Tools ASO (Allele Specific Oligonucleotides) –Good Knowledge of the allele to be tested is required for distinguishing one allele from another –Usually used in conjunction with PCR, electrophoresis and DNA blotting Other technologies for detecting specific point mutations are available –All suffer from the disadvantage that each test is directed toward a specific allele. A person affected by a different variant allele will not be detected as being affected.

5. DNA-based Tools DNA Sequencing Good for: –Idnetifying uncommon mutations Disadvantages –Phenotype may not be apparent from genotype –Mutations in promotor or introns that affect gene expression may be missed

6. DNA-based Tools DNA Chips Primarily a research tool for discovery of disease genes.

9. Protein-based Tools Western Blots Good for: –detecting the presence of a protein (e.g. Antitrypsin III) –detecting the presence of an antibody Disadvantages: –Can not determine if protein is active or not (e.g. antitrypsin III) –Must have access to tissue where protein is expressed (e.g. invasive biopsy might be required)

10. Protein-based Tools Immuno-histochemistry Good for: –Identifying tissues that express a particular protein –Showing over- or under- expression of a protein in a tissue (e.g. her-2 protein in breast cancer biopsy) Disadvantages: –Can not determine if protein is active or not (e.g. antitrypsin III) –Must have access to tissue where protein is expressed (e.g. invasive biopsy might be required)

11. Protein-based Tools Enzyme Assay Good for: –determining the activity of a suspect enzyme –determining the concentration of a metabolite Disadvantages: –Must have access to tissue where protein is expressed (e.g. invasive biopsy might be required) –Protein must be an enzyme or influence an enzyme activity –Activity must be stable during isolation of tissue

12. DNA-based Tools FISH (Fluorescence In Situ Hybridization) Good for: – visualization of large chromosomal changes Disadvantages: –Can not be used to detect small genetic changes

13. DNA-based Tools PCR Good for: –amplifying the amount of DNA. –isolating a segment of DNA Disadvantages: –Only semiquantitative – Linited size of segment to be amplified

14. DNA-based Tools PCR Examples: –Diagnosis of infectious disease (e.g. clamydia, gonorrhea, HIV) –Detection of a small deletion or insertion (e.g. the ΔF508 cystic fibrosis allele) –Amplification of a specific region to obtain DNA for other tests (e.g. ASO testing)

15. DNA-based Tools ASO (Allele Specific Oligonucleotides) Good for: – Distinguishing between known alleles that have small differences in DNA sequence (e.g. point mutations, small deletions or insertions) Disadvantages: – One needs to know the DNA sequences of the normal DNA and of the allele to be tested.

16. DNA-based Tools DNA sequencing Good for: –Idnetifying uncommon mutations Disadvantages –Phenotype may not be apparent from genotype –Mutations in promotor or introns that affect gene expression may be missed

17. DNA-based Tools DNA Chips Currently a research tool for discovery of disease genes. A potentially powerful diagnostic tool

18. Nucleic acid Basics Hybridization Electrophoresis PCR Diagnostic tools DNA-Protein interactions Chromatin Gene expression

19. Most Proteins bind in the Major Groove of DNA Major groove is visible here (minor groove would be seen from the back of the slide) Minor groove is visible here (major groove would be seen from the back of the slide)

20. Important functional groups located in the major groove H  Hydrogen bond acceptor H  Hydrogen bond donor VdW VanderWaals interaction Note the positions of the glycosidic bonds with respect to the major and minor grooves HH HH HH HH HH HH

21. Examples of Amino acid-Base Interactions

22. Common DNA-binding motifs Fig. 16.6 from Mark’s Basic Medical Biochemistry (page 288)

23. Common DNA-binding motifs Specific Examples The examples presented can be viewed via the “DNA-protein interactions” tutorial on the Molecular Basis of Medicine Web site