1. SNPs, Inheritance, and the Evolution of Lactose Tolerance
Got Milk?
SNPs, Inheritance, and the Evolution of Lactose Tolerance

2. Lactose Intolerance?

3. What’s Happening
Digestive System

4. 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
Students should read the background information for the lab at this point.
What is an SNP? Video

5. The Genetics The individual is lactose TOLERANT T
Chromosome 2 from dad T
LCT T
Chromosome 2 from mom
LCT
The individual is lactose TOLERANT

6. The Genetics The individual is lactose TOLERANT T
Chromosome 2 from dad T
LCT C
Chromosome 2 from mom
LCT
The individual is lactose TOLERANT

7. The Genetics The individual is lactose INTOLERANT C
Chromosome 2 from dad C
LCT C
Chromosome 2 from mom
LCT
The individual is lactose INTOLERANT

8. Where is it most prevalent?
Prevalence of lactose tolerance and reliance on dairy products vary throughout the world.

9. Why do we see this pattern?
Map shows % intolerance

10. Natural Selection There is variation of traits in a population
2. There is differential reproduction.
There is heredity.
End result.

11. 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

12. DNA consists of building blocks called nucleotides
What is DNA Made Of?
DNA consists of building blocks called nucleotides
Image: SCFBIO
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 atoms
variable

13. 2 Types of Nitrogenous Bases (4 in total)
Pyrimidines: 6-member ring
Cytosine & Thymine
Purines: fused 5 & 6 member rings
Adenine & Guanine
A T C G =
DNA alphabet

14. The Base Pair Rules 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

15. DNA has two strands with bases paired in the middle

16. DNA Replication
Molecular Cell Biology, Lodish et. al. 4th ed.

17. Deoxynucleotide-triphosphates: dNTPs
The “PCR Building Blocks”
A “dNTP mix” contains equal amounts of :
dATP
dTTP
dGTP
dCTP 11

18. The PCR “Cycle” Separates double helix into two strands
Denature: 94-96oC…
Anneal: oC…
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 3

19. After 30 cycles, DNA is amplified over a billion-fold!
Target sequence
Cycle 2
Cycle 1

20. PCR vs. Cellular DNA Replication

21. Tetra-primer ARMS-PCR Procedure for SNPs
Tetra-primer ARMS-PCR is a widely accepted method for amplifying SNP DNA. Unlike the traditional PCR procedure, it employs two sets of primers. The “outer primers” set the boundary for the DNA region containing the SNP. The “inner primers” are specific for each allele. During the PCR procedure, you will obtain either two or three different amplified DNA segments, depending upon genotype.
When the tetra-primer ARMS-PCR procedure is completed, the DNA fragments are separated by gel electrophoresis. The PCR products produce a characteristic fragment size pattern: Heterozygous individuals have one non-specific band and two specific bands of known length (3 bands total). Homozygotes have one non-specific and one specific band (two bands total).
A non-specific DNA fragment is produced in all all individuals. This DNA fragment results from the outer primers and serves as an internal positive control for the PCR procedure.
Homozygotes will produce one additional fragment resulting from one of the inner primers.
Heterozygotes will produce two additional DNA fragments, one from each of the inner primers.
The inner and outer primer arrangement for one particular SNP is shown below. The outer primer pair allows for the non-specific band to be produced. Each inner primer (G or A) is allele-specific, and individuals will produce that DNA segment only if they carry the specific allele.

22. Agarose Gel Electrophoresis

23. Agarose Gel Electrophoresis
DNA
“Ladder”
well #1 #2 #3 + _
large DNA
fragments
small DNA
Dye is added to
give the DNA color
Electron micrograph of an agarose gel

24. 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!

25. Sample Gel: Focus on the “lac” Lane
This gel shows some of the issues that might be seen with use of the tetra-primer system

26. How to Interpret the Gel
Extraneous bands at 400, 500
Outer band- 268
Inner band: 188
Inner band: 135 (faint)
Primer dimers <100

27. Credits