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From Genotype to Phenotype
PTC Taster Lab October 2015
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Bringing DNA science to more places
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PCR is at the heart of DNA analysis
Molecular diagnostics Text Consumer genomics Text Text Personalized medicine PCR Food and agriculture Text Text Human evolution Text Forensics
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PTC Taster Lab Links genotype and phenotype
Molecular physiology and genetics of taste Teach essential molecular biology techniques Aligns with standards
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The PTC lab packs a punch
Students use essential molecular biology techniques to determine genotype DNA extraction PCR Restriction digest Gel electrophoresis First class Second class Complete the miniPCR PTC Taster lab in two 45-min class periods, or in a single 3-h instruction block
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Experimental Outline What you’ll need: miniPCR
Bluegel electrophoresis system Pipettes, tips and tubes miniPCR PTC lab reagents kit Electrophoresis reagents Please refer to the Teacher’s Guide for details
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Sense of Taste Cheek cells divide very rapidly.
66 Cheek cells divide very rapidly. Transition: The various membranes in the cell provide a barrier between the cell’s environment and the cytoplasm. In order to retrieve the DNA the membranes have to be broken apart. Sense of Taste
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ORGAN Tongue CELL Gustatory TISSUE Papillae papillae taste buds
gustatory cells nerves
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PROTEIN: G-protein coupled receptor bitter taste receptors
----- Meeting Notes (10/20/15 14:20) ----- G Protein receptors make up 50% of drug targets for medications
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Each human carries a distinctive set of taste receptors which gives them a unique perception of how foods taste Taste is a phenotype Small differences in genotype can change taste perception
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Ability to taste PTC is Mendelian The TAS2R38 gene
Position (bp) Taster DNA aa non-Taster DNA aa 145 C Pro (P) G Ala (A) 785 T Val (V) 886 A Ile (I) Single nucleotide polymorphisms (SNPs) Genotype Phenotype Homozygous Taster PAV/PAV Intensely bitter Heterozygous Taster PAV/AVI Somewhat bitter Non-taster AVI/AVI Can’t taste
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Ability to taste PTC is Mendelian The TAS2R38 gene
Single nucleotide polymorphisms (SNPs) SNP1 (145) SNP2 (785) SNP3 (886) taster DNA C G aa P (Pro) A (Ala) V (Val) non-taster T A I (Ile) Genotype Phenotype Homozygous Taster PAV/PAV Intensely bitter Heterozygous Taster PAV/AVI Somewhat bitter Non-taster AVI/AVI Can’t taste
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Now, let’s try the TASTE test (PTC paper)
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Central Dogma…Genotype to Phenotype
Differences in DNA = genotype ‘taster’ allele ‘non-taster’ allele DNA TRANSCRIPTION ‘taster’ RNA ‘non-taster’ RNA AAAAAA RNA TRANSLATION Try slide with three columns, one for each genotype ----- Meeting Notes (10/21/15 17:18) ----- i need your help here zeke ‘taster’ protein ‘non-taster’ protein Protein SIGNAL TRANSDUCTION Differences in taste = phenotype
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Extract DNA From Cheek Cells
Cheek cells divide very rapidly. Transition: The various membranes in the cell provide a barrier between the cell’s environment and the cytoplasm. In order to retrieve the DNA the membranes have to be broken apart. Extract DNA From Cheek Cells Rub gently 5-6 times with a flat-head toothpick
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Use miniPCR as a Heat Block
DNA extraction PCR Restriction digest Gel electrophoresis Incubate 10 minutes at 95°C in miniPCR
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Using Windows app: 95C, 10 minutes
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Next step: PCR Amplify TAS2R38 gene around SNP785
DNA extraction PCR Restriction digest Gel electrophoresis Exponential amplification of the TAS2R38 gene
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Polymerase Chain Reaction (PCR)
Complex DNA sample Region of interest Amplified DNA (Billions of copies) PCR is used to replicate DNA outside the body
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PCR: exponential amplification
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Setting up a PCR experiment…
Template DNA to be amplified Pair of DNA primers DNA polymerase dNTPs Buffer to maintain pH and provide Mg2+ Thermal cycler dCTP dCTP dGTP Taq dATP dTTP primer A T G C dTTP dGTP dTTP dATP dCTP dGTP dATP
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How does PCR work? Repeat x 30 cycles Denaturation Annealing Extension
denatured DNA DNA + primers DNA + copy Single molecule 94° C ~1B copies 50-60° C 72° C Denaturation Annealing Extension Repeat x 30 cycles
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Programming the miniPCR…
1 2 3 4 5 6 ----- Meeting Notes (10/21/15 16:56) ----- have easel paper in TWO places around the room with BIG BOLD lettering to match the program requirements...
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Possible stopping point after PCR complete
DNA extraction PCR Store PCR product up to 48h at room temperature, or long term in fridge or freezer
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Next step: restriction digest
Interrogate SNP785 taster vs. non-taster variants DNA extraction PCR Restriction digest Gel electrophoresis Second class Beginning of second class period
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The restriction enzyme Fnu4H1 cuts @ SNP785 Only in the taster allele TAS2R38 variants
TAS2R38 Gene taster variant non-taster variant 5’…GCNGC…3’ 3’…CGNCG…5’ …TGTGCTGCCTT… …ACACGACGGAA… …TGTGTTGCCTT… …ACACAACGGAA… ----- Meeting Notes (10/21/15 16:55) ----- Zeke...can you work your magic with the taster SNP? I can't seem to get the cut right!!! RE …TGTGC TGCCTT… …ACACGA CGGAA… …TGTGTTGCCTT… …ACACAACGGAA…
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blueGel reduces equipment needs
DNA extraction PCR Restriction digest Gel electrophoresis No need for… big bulky gel chambers, power supplies, illuminators, or UV
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Homozygous dominant (taster)
Expected results… Homozygous dominant (taster) Homozygous recessive (non-taster) Heterozygous (taster) 300bp 200bp 100bp
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We hope you’ve enjoyed the lab
We hope you’ve enjoyed the lab! Now you can connect phenotype to genotype in two 45- minutes classes DNA extraction PCR Restriction digest Electrophoresis
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Contact miniPCR with feedback or questions
@miniPCR Facebook.com/miniPCR
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miniPCR™ Powerful Portable Engaging Affordable
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21st century DNA gel electrophoresis
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