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

17

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

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

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

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

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

Digest with HaeIII (GGCC)

-----GGCGGGCACT-------GGCGG CCACT----

Carolina Protocol - Page 10 Digest PCR products with HaeIII

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

Gel Electrophoresis
Gel electrophoresis

Gel Electrophoresis

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

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Carolina Protocol - Page 18

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Phenotype
Nontaster Genotype TT (homozygous) Tt (heterozygous) tt (homozygous) 4 1 1 0 1 6 0 Strong taster Weak taster

AA position QuickTime and a TIFF (LZW) decompressor 49 are neede d to see this picture. 262 296

Genotype association with taste phenotype Genotype Haplotype */PAV (*/T) No. of Subjects Sample Nontasters Utah NIH 3 1 38 21

Tasters 108 58 14 0

AVI/AVI (t/t) Utah NIH

*Any haplotype found in the sample.

Science. Volume 299 p. 1221 - 1225. 2003.

Why not exact match between phenotype and genotype?

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

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

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

Strong vs. Weak Tasters PTC threshold scores (higher score = better at tasting PTC) PAV/PAV (T/T) Utah 10.69 NIH 10.00 Utah 9.65 NIH 8.81 Utah 4.31 NIH 1.86

PAV/* (T/*)

AVI/AVI (t/t) *Any other haplotype

Science. Volume 299 p. 1221 - 1225. 2003.

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

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

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

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)

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

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

Possible risk/benefits for tasters?

May avoid green leafy vegetables and other strongly Flavored foods
Avoid plant poisons - a good thing!

Frequency of PTC gene haplotype in populations worldwide

Europe W. Asia E. Asia Africa SW Native American n=18 100%

Haplotype
PAV AVI AAV AAI

n=200
49% 47% 3%

n=22
33% 67%

n=54
69% 31%

n=24
50% 25% 4% 17%

PVI

4%

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.

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

Ala 49 (t/t)

Utah NIH

PAV = taster

AVI = Nontaster