Got Milk?

1. 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 What is an SNP? Video

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

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

7. The Genetics C Chromosome 2 from dad 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. 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 Image: SCFBIO http://www.scfbio-iitd.res.in/tutorial/gene.html What is DNA Made Of? DNA consists of building blocks called nucleotides

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 http://www.uic.edu/classes/bios/bios100/lectf03am/lect02.htm

14. The Base Pair Rules • Hydrogen bonds form between bases • A = Ttwo hydrogen bonds • G  Cthree 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

18. 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

19. Cycle 3 Cycle 2 Cycle 1 After 30 cycles, DNA is amplified over a billion-fold! Target sequence

20. Cellular DNA Replication PCR vs. Cellular DNA Replication

21. Tetra-primer ARMS-PCR Procedure for SNPs

22. Agarose Gel Electrophoresis

23. + DNA “Ladder” well #1 well #2 well #3 large DNA fragments small DNA fragments _ Agarose Gel Electrophoresis 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

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 http://cmgm.stanford.edu/~kimlab/www.babec.org http://www.dnalc.org/ http://evolution.berkeley.edu/