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PTC: DNA Analysis PCR amplified 221 base pairs of DNA from the PTC taste receptor gene. PTC: DNA Analysis PCR amplified 221 base pairs of DNA from the PTC taste receptor gene. PCR product Nontaster (t) Taster (T) -----GGCGGGCACT---(PCR product 221 bp) ----GGCGGCCACT---(PCR product 221 bp) PTC: DNA Analysis PCR amplified 221 base pairs of DNA from the PTC taste receptor gene. PCR product Nontaster (t) Taster (T) -----GGCGGGCACT---(PCR product 221 bp) ----GGCGGCCACT---(PCR product 221 bp) How can we analyze this DNA? 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* Haemophilus aegyptius 5'GGCC 3'CCGG 5'---GG CC---3' 3'---CC GG---5’ PCR product Nontaster (tt) Taster (TT) -----GGCGGGCACT---(PCR product 221 bp) ----GGCGGCCACT---(PCR product 221 bp) PCR product Nontaster (t) Taster (T) -----GGCGGGCACT---(PCR product 221 bp) ----GGCGGCCACT---(PCR product 221 bp) Digest with HaeIII (GGCC) -----GGCGGGCACT---- ----GGCGG CCACT---- PCR product Nontaster (tt) Taster (TT) -----GGCGGGCACT---(PCR product 221 bp) ----GGCGGCCACT---(PCR product 221 bp) Digest with HaeIII (GGCC) -----GGCGGGCACT---- ----GGCGG CCACT---- 221 base pair fragment 177 base pair fragment 44 base pair fragment 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 Protocol - Page 18 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Phenotype Nontaster Genotype TT (homozygous) Tt (heterozygous) tt (homozygous) T= PAV - Taster t = AVI - non-taster Strong taster Weak taster Genotype association with taste phenotype Genotype No. of Subjects Sample Nontasters Tasters */T Utah NIH 3 1 108 58 t/t Utah NIH 38 21 14 0 T= PAV - Taster t = AVI - non-taster 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 Other haplotypes are possible. 3% of Europeans have AAV haplotype. 48% of those with AAV are tasters t = AVI = non taster T = PAV = taster 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 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 Strong vs. Weak Tasters PTC threshold scores (higher score = better at tasting PTC) (T/T) Utah 10.69 NIH 10.00 (T/*) Utah 9.65 NIH 8.81 (t/t) Utah 4.31 NIH 1.86 *Any other haplotype Science. Volume 299 p. 1221 - 1225. 2003. To taste or not to taste? Have modern humans evolved to taste bitter compounds or not to taste bitter compounds? 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. The 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. 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. 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 Possible risks for non-tasters? Possible risks for non-tasters? Potential to eat toxic compounds. 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. Possible risks/benefits for tasters? Possible risks/benefits for tasters? May avoid green leafy vegetables and other healthy, but bitter foods Avoid plant poisons - a good thing! Frequency of PTC gene haplotype in populations worldwide Europe W. Asia E. Asia Africa Haplotype n=200 n=22 n=54 n=24 SW Native American n=18 PAV (T) 49% 33% 69% 50% 100% AVI (t) 47% 67% 31% 25% AAV 3% 4% AAI 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. PTC taste receptor structure QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Effect of homozygosity for Ala 49 (t/t) on phenotype. Genotype Ala 49 (t/t) PAV = taster Phenotype Sample No. of subjects (total no.) Nontaster Taster Utah NIH 48(51) 22(23) AVI = Nontaster 21(129) 3(61)