Lecture 5

• Assignment 1

• Polymorphic markers

• MusY marker

• Polyacrylamide electrophoresis

• Article 2 discussion

Assignment 1

Would you like the due date for the assignment moved to Oct 27, 2008?

The sequence marked with an X is the one that you are to analyze.

If you have another sequence file that does not have an X in its name do not analyze for the assignment!!!!!!!!!!!!!!

Example sequence

GGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCGAAGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGCCACCCGAAAAATACAGTCTTACTGGGCACAGAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTATCTGCCAGCGAAGATGCCACTATCAAGGTG

TGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGACAGCGTGCAGGACGTTTCCTTCGACGTCTCCGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGGACTTTCACCAGTCATTCGCCTGCGTGAAAACCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAAGGGGATTTCGTAGTGAGCGCCTCTAGGGATAAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTTAACGGGGCACAGAGAATGGGTACGGATGGCCAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAACAGTACGGGTTTGGCACGTGGCAACAAAGGAAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCATGGGCACCGGACAGTGCAAGAGCATCGATCAACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCTCGCATCTGGCTCGCGAGACAAAGTAATTCGTGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACGACAACTGGGTTCGCGGCATCGTCTTCCATCCTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTATNGGANACGCGCAACANANGGGTAATGAAA

ACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGCCATCCTTACGTGGTCNCCGGTAGTG

Step into liquid

Taken from Vallee et al., 2001

Taken from Tarricone et al., Neuron 2004

Mouse Murder Mystery 5Greta’s last case

DNA isolation

• Why can we isolate ancient DNA but not RNA?

• What should a good DNA isolation protocol do?

• What will the agarose gel in experiment 2 tell us?

Isolation of Genomic DNA using Commercial DNAzol

DNAzol

Homogenize

100%Ethanol

DNA

75%Ethanol

Precipitate Wash X2

dH2O

You will be assigned primer pairs individually for Experiment 2

These are your primers no help may be given to you by yourgroup mates.

Look up your assigned primer pair before arriving in the lab!You are expected to have worked out the master mix beforeyou arrive. The TA will check for this.

The assigned primer pairs are posted on the web.

What do the primers detect?

• 3G5, 5G4, and 6G2 detect autosomal DNA polymorphisms.

• MusY detects Y chromosome DNA.

List of primers for PCR

Name Primer Name Primer

3G5 For TGTCCCTGCTCAGAGAACAGAT

6G2 For TTCTTCACCTGCCTTCTTCCAC

3G5 Rev AAACACGATAA CACTGGGGG

6G2 Rev CCCTTTGCTTACCCAAGTTGCT

5G4 For AGTAGGCAGATAAGGGGTTTCC

MusY For TCCTTGGGCTCTTCATTATTCTTAAC

5G4 Rev TACAGCATCTAGTGAATGGGGG

MusY Rev GAGAACCACGTTGGTTTGAGATG

What is a DNA polymorphism?

How are you going to use DNA polymorphisms in this

experiment?

5G4

1 2 3 4 5 6 7

6G2

1 2 3 4 5 6 7

Example from previous Year

What do we need to know about the DNA polymorphisms to have an effective analysis?

What does it mean when we get a PCR product with MusY

primers?

Androgen insensitivity syndrome

Polyacylamide gel electrophoresis

Polyacrylamide Gel ElectrophoresisTo separate PCR products differing in only a few bp in length (for example, microsatellite markers), 6-10% PA gels are used.

Mixture of DNA molecules

Step by Step Instructions on how to assemble the polyacrylamide gel apparatus is posted on the course website

The TAs will be providing a demonstration in this week’s lab

Taken from Morrison and Boyd 3rd ed.

So what do we need to spot when looking at the

procedure?

• What molecule has the double bond.

• What produces the free radicals.

The reaction

Component Volume30% acrylamide/bis 4ml5X TBE 3ml10% APS 45μltemed 15μlwater 7.9total 15ml

Questions

• What has the double bonds?

• What is the function of bis acrylamide?

• What is the source of free radicals?

• What does temed do?

How the gel is made

Paper 2

The antiobesity gene

Obesity rates and calories available

Marion Nestle Scientific American Vol 297 2007

Supermarkets Ground Zero

Reagan in the early 1980s deregulated agriculture.

Farmers grow more food due to competitive forces.

The “shareholder value movement” of early 1980s.

Forced food companies to expand sales promoting:Between meals snackingLarger portionsEating in Book and Clothing stores.These were once taboo.

Marion Nestle Scientific American Vol 297 2007

Supermarkets Ground Zero

On entering a store see something colourful, aromatic and enticing.

Aisles and aisle-ends jam-packed with products: impulse buying.

Food companies pay supermarkets for prominent positions and large displays.

Checkout lines plastered with candy and other junk food items.

Marion Nestle Scientific American Vol 297 2007

1,000 calories: 59 sugar cubes

Marion Nestle Scientific American Vol 297 2007

Global obesity and global famine

Scientific American Vol 297 2007

Mexico’s problem living next to Coca Cola nation

Scientific American Vol 297 2007

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