got milk? snps, inheritance, and the evolution of lactose tolerance
TRANSCRIPT
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Got Milk?
SNPs, Inheritance, and the Evolution of Lactose Tolerance
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Lactose Intolerance?
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What’s Happening
Digestive System
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The Genetics• Tolerance is a mutation in
the LCT gene (Chromosome 2)
• Everyone has two copes of each gene (maternal and paternal)• Each gene has a C or a T at a
specific location– This is called a SNP (single
nucleotide polymorphism)– TT and TC = tolerant– CC = intolerant
What is an SNP? Video
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The Genetics
Chromosome 2 from dad
Chromosome 2 from mom
LCTLCT
LCTLCT
T
T
The individual is lactose TOLERANT
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The Genetics
Chromosome 2 from dad
Chromosome 2 from mom
LCTLCT
LCTLCT
T
C
The individual is lactose TOLERANT
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The Genetics
Chromosome 2 from dad
Chromosome 2 from mom
LCTLCT
LCTLCT
C
C
The individual is lactose INTOLERANT
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Where is it most prevalent?
Prevalence of lactose tolerance and reliance on dairy products vary throughout the world.
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Why do we see this pattern?
Map shows % intolerance
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Natural Selection
1. There is variation of traits in a population
2. There is differential reproduction.
3. There is heredity.
3. End result.
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How can we see this in our genes?
• PCR (polymerase chain reaction– Build copies of the segment
that contains the SNP so we can see it
• What’s in the mix?– Master Mix: Taq, dNTPs,
buffer– Primer Mix: 4 primers (two
outer primers and two inner primers)
PCR Reaction Video
The inner primers tell you your
genotype
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DNA consists of building blocks called nucleotides
Image: SCFBIOhttp://www.scfbio-iitd.res.in/tutorial/gene.html
1) a phosphate molecule gives DNA its negative charge
2) a pentose sugar five-carbon sugar in ring form
3) a nitrogenous base ring of carbon and nitrogen atomsvariable
What is DNA Made Of?
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Purines: fused 5 & 6 member ringsAdenine & Guanine
Pyrimidines: 6-member ring Cytosine & Thymine
http://www.uic.edu/classes/bios/bios100/lectf03am/lect02.htm
2 Types of Nitrogenous Bases (4 in total)
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Hydrogen bonds form between bases
A = T two hydrogen bonds
G C three hydrogen bonds
The bonds are weak and can be
broken by high temperatures
The Base Pair Rules
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DNA has two strands with bases paired in the middle
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Molecular Cell Biology, Lodish et. al. 4th ed.
DNA Replication
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The “PCR Building Blocks”
A “dNTP mix” contains equal amounts of :
dATP
dTTP
dGTP
dCTP
Deoxynucleotide-triphosphates: dNTPs
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The PCR “Cycle”
Denature: 94-96oC…
Anneal: 37-65oC…
Extension: 72oC…
Repeat steps 1-3:
Separates double helix into two strands
Primers bind to target site on single stranded DNA
DNA polymerase adds dNTPs according to the base pairing rules (polymerization)
5 to 40 times using a Thermal Cycler
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Cycle 1
Cycle 2
Cycle 3
After 30 cycles, DNA is amplified over a billion-fold!
Cycle Relative Amount 1 22 43 84 165 326 647 1288 2569 512
10 1,024. .. .
20 1,048,576. .. .
30 1,073,741,824
Target sequence
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Polymerase Chain Reaction
Cell
What are we copying?
How do we separate the DNA?
What is doing the copying?
How do we fish out the sequence?
What does the work?
Cellular DNA Replication
PCR vs. Cellular DNA Replication
DNA DNA
Heat Enzymes
Taq polymerase Human polymerase
Primers Primers
Thermal cycler Cell
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Tetra-primer ARMS-PCR Procedure for SNPs
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Agarose Gel Electrophoresis
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Dye is added to give the DNA color
DNA “Ladder”
well#1
well #2
well #3
+_
large DNAfragments
small DNAfragments
Electron micrograph of an agarose gel
Agarose Gel Electrophoresis
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The Tetra-Primer System
• It’s not super “clean”• You get primer dimers
• Inner primers are similar and stick together • Ignore them on the gel (they’re smaller
than 100 bp)• The outer primers are sometimes non-specific• The fragments are small! Run the gel for the full
time to separate out the bands!
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Sample Gel: Focus on the “lac” Lane
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How to Interpret the Gel
Primer dimers <100
Inner band: 135 (faint)
Inner band: 188
Outer band- 268
Extraneous bands at 400, 500
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Credits
http://cmgm.stanford.edu/~kimlab/www.babec.orghttp://www.dnalc.org/
http://evolution.berkeley.edu/