From Genotype to Phenotype PTC Taster Lab October 2015

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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

PTC Taster Lab Links genotype and phenotype Molecular physiology and genetics of taste Teach essential molecular biology techniques Aligns with standards

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

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

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

ORGAN Tongue CELL Gustatory TISSUE Papillae papillae taste buds gustatory cells nerves

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

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

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

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

Now, let’s try the TASTE test (PTC paper)

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

Extract DNA From Cheek Cells 14141414 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

Use miniPCR as a Heat Block DNA extraction PCR Restriction digest Gel electrophoresis Incubate 10 minutes at 95°C in miniPCR

Using Windows app: 95C, 10 minutes

Next step: PCR Amplify TAS2R38 gene around SNP785 DNA extraction PCR Restriction digest Gel electrophoresis Exponential amplification of the TAS2R38 gene

Polymerase Chain Reaction (PCR) Complex DNA sample Region of interest Amplified DNA (Billions of copies) PCR is used to replicate DNA outside the body

PCR: exponential amplification

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

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

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...

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

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

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…

blueGel reduces equipment needs DNA extraction PCR Restriction digest Gel electrophoresis No need for… big bulky gel chambers, power supplies, illuminators, or UV

Homozygous dominant (taster) Expected results… Homozygous dominant (taster) Homozygous recessive (non-taster) Heterozygous (taster) 300bp 200bp 100bp

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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