ptc: dna analysis pcr amplified 221 base pairs of dna from the ptc taste receptor gene
DESCRIPTION
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) -----GGC GGGC ACT--------GGC GGCC ACT---- - PowerPoint PPT PresentationTRANSCRIPT
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---- ----GGCGGCCACT----(PCR product 221 bp) (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---- ----GGCGGCCACT----(PCR product 221 bp) (PCR product 221 bp)
How can we analyze this DNA?
• 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'GGCC3'CCGG
5'---GG CC---3'3'---CC GG---5’
Restriction Enzymes
PCR product
Nontaster (tt) Taster (TT)
-----GGCGGGCACT---- ----GGCGGCCACT----(PCR product 221 bp) (PCR product 221 bp)
PCR product
Nontaster (t) Taster (T)
-----GGCGGGCACT---- ----GGCGGCCACT----(PCR product 221 bp) (PCR product 221 bp)
Digest with HaeIII (GGCC)
-----GGCGGGCACT---- ----GGCGG CCACT----
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
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
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Carolina Protocol - Page 18
Phenotype
Nontaster Strong taster Weak tasterGenotype
TT (homozygous)
Tt (heterozygous)
tt (homozygous)
T= PAV - Tastert = AVI - non-taster
Genotype association with taste phenotype
Genotype No. of SubjectsSample Nontasters Tasters
*/T Utah 3 108NIH 1 58
t/t Utah 38 14NIH 21 0
T= PAV - Tastert = 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 tasterT = 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 10.69NIH 10.00
(T/*) Utah 9.65NIH 8.81
(t/t) Utah 4.31NIH 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 ornot 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 leafyvegetables (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, butbitter foods
Avoid plant poisons - a good thing!
Non taster AVI observed in all populations except Southwest Native Americans who are exclusively homozygous for PAV. Founder populations move out of AfricaScience. Volume 299 p. 1221 - 1225. 2003.
Frequency of PTC gene haplotype in populations worldwide
Europe W. Asia E. Asia Africa SW Native American
Haplotype n=200 n=22 n=54 n=24 n=18
PAV (T) 49% 33% 69% 50% 100%
AVI (t) 47% 67% 31% 25%
AAV 3% 4%
AAI 17%
PVI 4%
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PTC taste receptor structure
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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