1. PTC: DNA Analysis
PCR amplified 221 base pairs of DNA from the PTC
taste receptor gene.

2. PTC: DNA Analysis
PCR amplified 221 base pairs of DNA from the PTC
taste receptor gene.
PCR product
Nontaster (t) Taster (T)
-----GGCGGGCACT GGCGGCCACT----
(PCR product 221 bp) (PCR product 221 bp)

3. PTC: DNA Analysis
PCR amplified 221 base pairs of DNA from the PTC
taste receptor gene.
PCR product
Nontaster (t) Taster (T)
-----GGCGGGCACT GGCGGCCACT----
(PCR product 221 bp) (PCR product 221 bp)
How can we analyze this DNA?

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

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

6. Restriction Enzymes
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

7. Restriction Enzymes HaeIII* Haemophilus aegyptius 5'GGCC 3'CCGG

8. PCR product
Nontaster (tt) Taster (TT)
-----GGCGGGCACT GGCGGCCACT----
(PCR product 221 bp) (PCR product 221 bp)

9. PCR product
Nontaster (t) Taster (T)
-----GGCGGGCACT GGCGGCCACT----
(PCR product 221 bp) (PCR product 221 bp)
Digest with HaeIII
(GGCC)
-----GGCGGGCACT GGCGG CCACT----

10. PCR product
Nontaster (tt) Taster (TT)
-----GGCGGGCACT GGCGGCCACT----
(PCR product 221 bp) (PCR product 221 bp)
Digest with HaeIII
(GGCC)
-----GGCGGGCACT GGCGG CCACT----
221 base pair fragment 177 base pair fragment
44 base pair fragment

11. 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
What is the charge on DNA and proteins? What is the shape? Why is this important?
How would you connect the electrodes?

12. Gel Electrophoresis
Gel electrophoresis

13. Gel Electrophoresis

14. Carolina Protocol - Page 18

15. Phenotype
Nontaster Strong taster Weak taster
Genotype
TT (homozygous)
Tt (heterozygous)
tt (homozygous)
T= PAV - Taster
t = AVI - non-taster

16. Genotype association with taste phenotype Genotype No. of Subjects
Sample Nontasters Tasters
*/T Utah
NIH
t/t Utah
NIH
T= PAV - Taster
t = AVI - non-taster
Science. Volume 299 p

17. Why not exact match between phenotype and genotype?

18. Why not exact match between phenotype and genotype?
Other nucleotide substitutions may be present

19. Why not exact match between phenotype and genotype?
Other nucleotide substitutions may be present
Other haplotypes are possible.
3% of Europeans have AAV haplotype.
48% of those with AAV are tasters
t = AVI = non taster
T = PAV = taster

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

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

22. PTC threshold scores (higher score = better at tasting PTC)
Strong vs. Weak Tasters
PTC threshold scores (higher score = better at tasting PTC)
(T/T) Utah 10.69
NIH
(T/*) Utah 9.65
NIH 8.81
(t/t) Utah 4.31
NIH 1.86
*Any other haplotype
Science. Volume 299 p

23. To taste or not to taste?
Have modern humans evolved to taste bitter compounds or
not to taste bitter compounds?

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

25. Why would we have evolved to lose the ability to taste PTC?
Heterozygotes might be able to taste a broader range of
bitter compounds. The non tasting allele may bind to different
bitter compounds.

26. Why would we have evolved to lose the ability to taste PTC?
Heterozygotes might be able to taste a broader range of
bitter compounds. The 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.

27. Why would we have evolved to lose the ability to taste PTC?
Heterozygotes might be able to taste a broader range of
bitter compounds. The 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.
More likely to consume varied diet that includes green leafy
vegetables (which contain thiocynates) which could offer
protection against thyroid disorders

28. Possible risks for non-tasters?

29. Possible risks for non-tasters?
Potential to eat toxic compounds.

30. Possible risks for non-tasters?
Potential to eat toxic compounds.
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.

31. Possible risks/benefits for tasters?

32. Possible risks/benefits for tasters?
May avoid green leafy vegetables and other healthy, but
bitter foods
Avoid plant poisons - a good thing!

33. Frequency of PTC gene haplotype in populations worldwide
Europe W. Asia E. Asia Africa SW Native
American
Haplotype n= n= n=54 n=24 n=18
PAV (T) 49% % % % %
AVI (t) 47% % % %
AAV % %
AAI %
PVI %
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

34. PTC taste receptor structure

36. Effect of homozygosity for Ala 49 (t/t) on phenotype.
Genotype Phenotype
Sample No. of subjects (total no.)
Nontaster Taster
Ala 49 (t/t) Utah 48(51) 21(129)
NIH 22(23) 3(61)
PAV = taster AVI = Nontaster