1. Carolina Biological Supply Company: Using a SNP to detect Bitter Tasting Ability: p. 17

2. Restriction enzymes were discovered in E.coli as a defense mechanism against bacterial viruses (bacteriophages) Restriction Enzymes

3. Restriction enzymes were discovered in E.coli as a defense mechanism against bacterial viruses (bacteriophages) They cut double stranded DNA at sequence specific sites Restriction Enzymes

4. Restriction enzymes were discovered in E.coli as a defense mechanism against bacterial viruses (bacteriophages) They cut double stranded DNA at sequence specific sites 1978 Nobel Prize in Medicine was awarded to Werner Arber, Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases DNAi Restriction Enzyme Restriction Enzymes

5. HaeIII* Haemophilus aegyptius 5'GGCC 3'CCGG 5'---GG CC---3' 3'---CC GG---5 ’ Restriction Enzymes

6. PCR product Nontaster (tt)Taster (TT) -----GGCGGGCACT--------GGCGGCCACT----(PCR product 221 bp) Digest with HaeIII (GGCC) -----GGCGGGCACT---- ----GGCGG CCACT---- Carolina Protocol - Page 10 Digest PCR products with HaeIII

7. Gel Electrophoresis Gel electrophoresis is used to separate nucleic acids (DNA and RNA) or proteins for analytical use –DNA and RNA are separated using agarose –The gel is a matrix (cross-linked polymers) that allow products to be separated Separation is based on the size (not shape) of a product as it moves through a charged field

8. Gel Electrophoresis Gel electrophoresis

9. Gel Electrophoresis

10. Carolina Biological Supply Company: Using a SNP to detect Bitter Tasting Ability: p. 17

11. Carolina Protocol - Page 18

12. Phenotype NontasterStrong taster Weak taster Genotype TT (homozygous)11 Tt (heterozygous)16 tt (homozygous)400 AA position 49 262 296

13. Genotype association with taste phenotype Genotype No. of Subjects HaplotypeSampleNontastersTasters */PAV (*/T)Utah3108 NIH158 AVI/AVI (t/t)Utah3814 NIH210 *Any haplotype found in the sample. Science. Volume 299 p. 1221 - 1225. 2003.

14. Why not exact match between phenotype and genotype?

15. Other nucleotide substitutions may be present

16. Why not exact match between phenotype and genotype? Other nucleotide substitutions may be present Genes on other chromosomes may influence ability to taste PTC

17. Why not exact match between phenotype and genotype? Other nucleotide substitutions may be present Genes on other chromosomes may influence ability to taste PTC PTC paper has extremely low concentration PTC

18. Strong vs. Weak Tasters PTC threshold scores (higher score = better at tasting PTC) PAV/PAV (T/T) Utah10.69 NIH 10.00 PAV/* (T/*)Utah9.65 NIH 8.81 AVI/AVI (t/t)Utah 4.31 NIH1.86 *Any other haplotype Science. Volume 299 p. 1221 - 1225. 2003.

19. Evolution of Taste: Have modern humans evolved to taste bitter compounds or not to taste bitter compounds?

20. Evolution of Taste: Have modern humans evolved to taste bitter compounds or not to taste bitter compounds? Can all non human primates detect PTC?

21. Why would we have evolved to lose the ability to taste PTC?

22. Heterozygotes might be able to taste a broader range of bitter compounds (non tasting allele may bind to different bitter compounds)

23. Why would we have evolved to lose the ability to taste PTC? Heterozygotes might be able to taste a broader range of bitter compounds (non tasting allele may bind to different bitter compounds) Populations with low sensitivity to PTC are found in areas harboring endemic malaria. Non tasters might be predisposed to consume plant-derived anti-malarial compounds

24. Possible risk/benefits for non-tasters? Studies suggest that nontasters, who are not aversive to the bitter taste of cigarettes, may be more at risk for heavy smoking and therefore more vulnerable to nicotine addiction. More likely to consume varied diet that includes green leafy vegetables (which contain thiocynates) which could offer protection against thyroid disorders

25. Possible risk/benefits for tasters? May avoid green leafy vegetables and other strongly Flavored foods Avoid plant poisons - a good thing!

26. Non taster AVI observed in all populations except Southwest Native Americans who are exclusively homozygous for PAV. Founder populations move out of Africa Science. Volume 299 p. 1221 - 1225. 2003. Frequency of PTC gene haplotype in populations worldwide Europe W. Asia E. AsiaAfrica SW Native American Haplotypen=200 n=22 n=54n=24 n=18 PAV 49% 33% 69% 50% 100% AVI 47% 67% 31% 25% AAV 3% 4% AAI17% PVI 4%

28. Effect of homozygosity for Ala 49 (t/t) on phenotype. GenotypePhenotype SampleNo. of subjects (total no.) NontasterTaster Ala 49 (t/t)Utah48(51)21(129) NIH22(23)3(61) PAV = tasterAVI = Nontaster