Download Prepractical demo_SF_Class_2009

1/31/11
Genetics module
Lectures: 5 x 4 = 20
  DNA Structure, Replication
  The Genetic Code; Transcription and Translation
  Principles of Heredity; Gene Mapping
  Controlling Gene Expression
  Mutation and Cancer
Textbook: Introduction to Genetic Analysis, Griffiths et al., 9th Ed.
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Practical schedule:
  Day 1: Isolating DNA: Dr. Kevin Mitchell
- Isolating DNA from our cheek Cells
- Polymerase Chain Reaction (PCR)
  Day 2: Comparing our genotype to our phenotype: Dr. Kevin Mitchell
- Restriction Enzyme Digestion
- Gel Electrophoresis
- PTC taste test
  Day 3: Bioinformatics - Analysing Sequences: Prof. Ken Wolfe
- DNA, RNA, Protein
  Day 4: Gene Expression 1: Dr. Frank Wellmer
- Comparing Gene Expression in Human Tissues
- Transgenic Reporter Genes
  Day 5: Gene Expression 2: Dr. Frank Wellmer
- Comparing Gene Expression in Human Tissues
- Transgenic Reporter Genes
Assessment
  Lab-book reports (30%)
  Multiple choice exam on Day 5 (70%)
Lab-book reports:
- Aim (major goal and experimental aims)
- Observations
- Discussion
Due on Days 2, 3, 5
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Genetic differences in taste perception
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Different tastes detected by different types of receptor proteins
Expressed in different cells in taste buds
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Taste receptor genes
  Single genes for sweet, umami, sour receptors
  Large family of bitter receptor genes
- different ones detect different chemicals
- all transmit same signal: “bitter”
  Species-specificity
- repertoire of receptors adapted for chemicals
that must be detected
e.g. cats lack functional sweet receptor
and do not prefer sweet-tasting foods
  Genetic variation within species
- mutations in taste receptor genes account for
some differences in people’s tastes
TAS2R38 receptor detects PTC
Phenylthiocarbamide
But only some people can taste PTC!
This difference is inherited
Due to mutations in TAS2R38 gene
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Cloning the PTC taste sensitivity gene
  Taster phenotype found to run in families
  Transmitted as a Mendelian trait
- one major gene involved
  Gene was mapped to chromosome 7
  TAS2R38 gene: encodes a bitter receptor protein
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Red: Taster sequence
Blue: Non-taster sequence
Two major alleles (variants) in human population:
PAV allele: ~55% (taster)
AVI allele: ~40% (non-taster)
Only need one functional (taster) allele to be able to taste PTC
What is expected frequency of tasters and non-tasters
in the population?
Can you think of another case where a mutation in a
receptor protein alters perception?
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Determining which TAS2R38 alleles you carry
(your genotype)
  Isolate some of your DNA
  How to analyse just the TAS2R38 gene?
(among 3x109 total bases of sequence)
  Use Polymerase Chain Reaction to amplify the
TAS2R38 gene sequence specifically
  How to tell which allele(s) you have?
  Use Restriction Enzyme that digests one allele
but not the other
Isolating your DNA
  Collect some cells:
- rinse mouth vigorously with saline to
collect cheek cells
- spin down saline to concentrate cells
  Burst them open:
- resuspend cells and add small amount
to tube containing Chelex resin (binds
heavy metal ions that could damage DNA)
- boil for ten minutes (bursts cells, degrades proteins)
  Purify the DNA away from everything else (proteins, lipids)
- spin down cell debris and resin
- carefully remove supernatant containing DNA
Make sure tubes are labeled with your ID number!
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The Polymerase Chain Reaction
  Selectively replicate specific sequence over and over
(amplification)
  DNA replication requires:
- DNA template
- DNA polymerase enzyme
- Nucleotides
- Primer
  Primer is a short stretch of DNA that binds to its
complementary sequence in the template and acts as
start of new strand of DNA
  Use primers complementary to sequence of interest
- amplify only that region of DNA
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PCR is hugely sensitive
Used in forensics to
amplify DNA from tiny
amounts of cellular
material
Amplify regions that are
highly variable in
population: “DNA
fingerprinting”
Very sensitive to
contamination!
Using Gilson pipettes
1.  Use appropriate pipette for volume in question
2.  Two-stop mechanism – first stop to suck up correct
amount; full depression to expel all liquid
3.  Be sure not to take extra liquid on outside of tip
4.  Be sure to change tips for every solution / tube
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Red: Taster sequence
Blue: Non-taster sequence
Amplifying TAS2R38 sequence from your purified DNA
  Put some of your purified DNA in PCR tube
  Use another tube for negative control (no DNA)
  Add primers,nucleotides, Taq DNA polymerase, buffer
  Mix together, store on ice, then place in thermal cycler
- goes through many rounds of temperature shifts
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We will use PCR to amplify region of TAS2R38 gene that has
polymorphism known to affect ability to taste PTC
End up with tube full of just this little stretch of DNA
How to tell which allele(s) are present?
1.  Could sequence the DNA
OR:
2. Use enzyme that can tell the difference
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Restriction Enzymes
  Derived from bacteria
- host defense versus viruses
  Restriction Enzymes cleave DNA at a specific sequence
(e.g., 5’ GAATTC 3’, 5’ CATATG 3’, 5’ CTGCAG 3’)
Can you see anything unusual about these sequences?
Why don’t they cleave the bacteria’s DNA?
  We want an enzyme that will cleave one allele of the
TAS2R38 sequence but not the other
- need polymorphism itself to be part of the cleavage site
HaeIII cleaves sequence: GGCC
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Gel Electrophoresis
How to tell if amplified DNA has been cut by restriction enzyme?
  Run it through a gel using an electric current
  Gel matrix retards passage of larger molecules more than
smaller ones
  Separate pieces of DNA by size - visualise with fluorescent
chemical label
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Homozygous Non-Taster
Heterozygous
Homozygous Taster
Control Lane
Testing for perception of PTC
Does your phenotype correlate with your genotype?
Does it correlate with whether you like brussel sprouts?
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Site GGCC/GGGC not actually there in the genome
(usual sequence either AGCC or AGGC)
We can change the A to a G in the amplified DNA by
incorporating a G at that position in the primer
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