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

Chromosome 2 from dad

Chromosome 2 from mom

LCTLCT

LCTLCT

T

T

The individual is lactose TOLERANT

The Genetics

Chromosome 2 from dad

Chromosome 2 from mom

LCTLCT

LCTLCT

T

C

The individual is lactose TOLERANT

The Genetics

Chromosome 2 from dad

Chromosome 2 from mom

LCTLCT

LCTLCT

C

C

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 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?

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)

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

DNA has two strands with bases paired in the middle

Molecular Cell Biology, Lodish et. al. 4th ed.

DNA Replication

The “PCR Building Blocks”

A “dNTP mix” contains equal amounts of :

dATP

dTTP

dGTP

dCTP

Deoxynucleotide-triphosphates: dNTPs

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

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

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

Tetra-primer ARMS-PCR Procedure for SNPs

Agarose Gel Electrophoresis

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

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

Primer dimers <100

Inner band: 135 (faint)

Inner band: 188

Outer band- 268

Extraneous bands at 400, 500

Credits

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

http://evolution.berkeley.edu/