Download PTC Lab Classroom Slides

From Genotype to Phenotype
PTC Taster Lab
October 2015
Bringing DNA science to more places
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PCR is at the heart of DNA analysis
Molecular
diagnostics
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Consumer
genomics
Personalized
medicine
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Food and
agriculture
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PCR
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Human
evolution
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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
First class
PCR
Restriction
digest
Gel
electrophoresis
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
papillae
taste buds
CELL
• Gustatory
TISSUE
• Papillae
gustatory cells
nerves
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bitter taste
receptors
PROTEIN:
G-protein
coupled receptor
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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
Single
nucleotide
polymorphisms
(SNPs)
Position
(bp)
Taster
DNA
aa
145
C
Pro (P)
G
Ala (A)
785
C
Ala (A)
T
Val (V)
886
G
Val (V)
A
Ile (I)
Genotype
Homozygous Taster
Heterozygous Taster
Non-taster
PAV/PAV
PAV/AVI
AVI/AVI
non-Taster
DNA
aa
Phenotype
Intensely bitter
Somewhat bitter
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)
DNA
C
C
G
aa
P (Pro)
A (Ala)
V (Val)
DNA
G
T
A
aa
A (Ala)
V (Val)
I (Ile)
taster
non-taster
Genotype
Homozygous Taster
Heterozygous Taster
Non-taster
PAV/PAV
PAV/AVI
AVI/AVI
Phenotype
Intensely bitter
Somewhat bitter
Can’t taste
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Central Dogma…Genotype to Phenotype
Differences in DNA = genotype
‘taster’ allele
‘non-taster’ allele
DNA
TRANSCRIPTION
AAAAAA
‘taster’ RNA
‘non-taster’ RNA
RNA
TRANSLATION
Protein
‘taster’ protein
‘non-taster’ protein
SIGNAL TRANSDUCTION
Differences in taste = phenotype
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Extract DNA From Cheek Cells
Rub gently 5-6
times with a flathead 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…
1. Template DNA to be amplified
2. Pair of DNA primers
3. DNA polymerase
Taq
ATP
d
primer
primer
4. dNTPs
G
5. Buffer to maintain pH and provide Mg2+
A
A
C TG
G
TA
C
C
CTP
d
6. Thermal cycler
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How does PCR work?
denatured DNA
Single molecule
DNA + primers
DNA + copy
94° C
72° C
~1B copies
50-60° C
Denaturation
Annealing
Extension
Repeat x 30 cycles
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Programming the miniPCR…
2
3
4
1
5
6
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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
SNP
TAS2R38 Gene
taster variant
…TGTG
TGCCTT…
…ACAC
ACGGAA…
C
…ACACGA
…TGTG
TGCCTT…
CGGAA…
non-taster variant
TTGCCTT…
…ACACAACGGAA…
…TGTG
TTGCCTT…
…ACACAACGGAA…
…TGTG
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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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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!
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
www.minipcr.com
@miniPCR
Facebook.com/miniPCR
[email protected]
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miniPCR™
Powerful
Portable
Engaging
Affordable
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21st century DNA gel electrophoresis
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