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

PCR

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Forensics

Food and

agriculture

Consumer

genomics

Molecular

diagnostics

Personalized

medicine

Human

evolution

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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

DNA extractionGel

electrophoresisPCR

Restriction

digest

Students use essential molecular biology techniques to determine genotype

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

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ORGAN

• Tongue

TISSUE

• Papillae

CELL

• Gustatory

taste buds

papillae

gustatory cells

nerves

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PROTEIN: G-protein

coupled receptor

bitter taste receptors

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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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Position

(bp)

Taster

DNA aa

non-Taster

DNA aa

145 C Pro (P) G Ala (A)

785 C Ala (A) T Val (V)

886 G Val (V) A Ile (I)

Homozygous Taster PAV/PAV Intensely bitterHeterozygous Taster PAV/AVI Somewhat bitterNon-taster AVI/AVI Can’t taste

Ability to taste PTC is Mendelian

The TAS2R38 gene

Genotype Phenotype

Single nucleotide

polymorphisms (SNPs)

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Single nucleotide polymorphisms (SNPs)

SNP1 (145) SNP2 (785) SNP3 (886)

taster

DNA C C G

aa P (Pro) A (Ala) V (Val)

non-taster

DNA G T A

aa A (Ala) V (Val) I (Ile)

Homozygous Taster PAV/PAV Intensely bitterHeterozygous Taster PAV/AVI Somewhat bitterNon-taster AVI/AVI Can’t taste

Ability to taste PTC is Mendelian

The TAS2R38 gene

Genotype Phenotype

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TRANSCRIPTION

TRANSLATION

SIGNAL TRANSDUCTION

AAAAAA

Central Dogma…Genotype to Phenotype

‘taster’ allele ‘non-taster’ allele

‘taster’ RNA ‘non-taster’ RNA

‘non-taster’ protein‘taster’ protein

Differences in DNA = genotype

Differences in taste = phenotype

DNA

RNA

Protein

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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 extractionGel

electrophoresisPCR

Restriction

digest

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 PCRGel

electrophoresis

Restriction

digest

Exponential amplification of

the TAS2R38 gene

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Polymerase Chain Reaction (PCR)

Complex DNA sample

Amplified DNA (Billions of copies)

Region ofinterest

PCR is used to replicate DNA outside the body

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PCR: exponential amplification

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1. Template DNA to be amplified

2. Pair of DNA primers

3. DNA polymerase

4. dNTPs

5. Buffer to maintain pH and provide Mg2+

6. Thermal cycler

primer

primer

Taq

AA

AT

T

C C C GG

G

dATP

dCTP

Setting up a PCR experiment…

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How does PCR work?

Denaturation Annealing Extension

DNA + primersdenatured DNA DNA + copy

94° C

50-60° C

72° C

Repeat x 30 cycles

Single molecule ~1B copies

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1

2

3

4

5

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Programming the miniPCR…

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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

DNA extractionGel

electrophoresisPCR

Restriction

digest

Interrogate SNP785 taster vs. non-taster variants

Second class

Beginning of second class period

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TAS2R38 Gene

SNP

taster variant non-taster variant

The restriction enzyme Fnu4H1 cuts @ SNP785 Only in the taster allele TAS2R38 variants

…TGTGTTGCCTT…

…ACACAACGGAA…

…TGTG TGCCTT…

…ACAC ACGGAA…

…TGTGTTGCCTT…

…ACACAACGGAA…

…TGTGC TGCCTT…

…ACACGA CGGAA…

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Restriction

digest

blueGel reduces equipment needs

DNA extraction PCRGel

electrophoresis

No need for… big bulky gel chambers, power supplies, illuminators, or UV

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Expected results…

Homozygous

dominant

(taster)

Homozygous

recessive

(non-taster)

Heterozygous

(taster)

100bp

200bp

300bp

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DNA extraction ElectrophoresisPCRRestriction

digest

We hope you’ve enjoyed the lab!Now you can connect phenotype to genotype in two 45- minutes classes

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Contact miniPCR with feedback or questions

www.minipcr.com

@miniPCR

Facebook.com/miniPCR

team@minipcr.com

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miniPCR™

Powerful

Portable

Engaging

Affordable

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21st century DNA gel electrophoresis