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Got Milk?
SNPs, Inheritance, and the Evolution
of Lactose Tolerance
Lactose Intolerance?
What’s Happening
Digestive System
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
The Genetics
T
Chromosome
2 from dad
LCT
Chromosome
2 from mom
T
LCT
The individual is lactose TOLERANT
The Genetics
T
Chromosome
2 from dad
LCT
Chromosome
2 from mom
C
LCT
The individual is lactose TOLERANT
The Genetics
C
Chromosome
2 from dad
LCT
Chromosome
2 from mom
C
LCT
The individual is lactose INTOLERANT
Where is it most prevalent?
Prevalence of lactose tolerance and reliance on dairy products vary throughout the world.
Why do we see this pattern?
Map shows % intolerance
Natural Selection
1. There is variation of traits in a
population
2. There is differential reproduction.
3. There is heredity.
3. End result.
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
DNA Replication
Molecular Cell Biology, Lodish et. al. 4th ed.
Deoxynucleotide-triphosphates: dNTPs
The “PCR Building Blocks”
A “dNTP mix” contains equal
amounts of :
dATP
dTTP
dGTP
dCTP
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
After 30 cycles, DNA is amplified over a billion-fold!
Cycle 3
Target sequence
Cycle 2
Cycle 1
Cycle
1
2
3
4
5
6
7
8
9
10
.
.
20
.
.
30
Relative Amount
2
4
8
16
32
64
128
256
512
1,024
.
.
1,048,576
.
.
1,073,741,824
PCR vs. Cellular DNA Replication
Polymerase Chain
Reaction
Cellular DNA
Cell
Replication
What are we copying?
DNA
DNA
How do we separate the DNA?
Heat
Enzymes
What is doing the copying?
Taq polymerase
Human polymerase
How do we fish out the sequence?
Primers
Primers
What does the work?
Thermal cycler
Cell
Tetra-primer ARMS-PCR Procedure
for SNPs
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
well
#1
well
#2
well
#3
_
DNA
“Ladder”
large DNA
fragments
small DNA
fragments
Dye is added to
give the DNA color
+
Electron micrograph of an agarose gel
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!
Sample Gel:
Focus on the “lac” Lane
How to Interpret the Gel
Extraneous bands at 400, 500
Outer band- 268
Inner band: 188
Inner band: 135 (faint)
Primer dimers <100