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
GGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCGA
AGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGCCACCCGAAAAATACAGTCTTACTGGGCACA
GAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTATCTGCCAGCGAAGATGCCACTATCAAGGTG
TGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGACAGCGTGCAGGACGTTTCCTTCGACGTCTC
CGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGGACTTTCACCAGTCATTCGCCTGCGTGAAAA
CCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAAGGGGATTTCGTAGTGAGCGCCTCTAGGGAT
AAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTTAACGGGGCACAGAGAATGGGTACGGATGGC
CAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAACAGTACGGGTTTGGCACGTGGCAACAAAGG
AAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCATGGGCACCGGACAGTGCAAGAGCATCGATC
AACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCTCGCATCTGGCTCGCGAGACAAAGTAATTCG
TGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACGACAACTGGGTTCGCGGCATCGTCTTCCATC
CTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTATNGGANACGCGCAACANANGGGTAATGAAA
ACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGCCATCCTTACGTGGTCNCCGGTAGTG
Step into liquid
Taken from Vallee et al., 2001
Taken from Tarricone
et al., Neuron 2004
Mouse Murder Mystery 5
Greta’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
100%
Ethanol
75%
Ethanol
Homogenize
Precipitate
Wash X2
dH2O
DNA
You will be assigned primer pairs individually for Experiment 2
These are your primers no help may be given to you by your
group mates.
Look up your assigned primer pair before arriving in the lab!
You are expected to have worked out the master mix before
you 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
TGTCCCTGCTCAG
AGAA CAGA T
6G2 For
TTCTTCACCTGCCT
TCTTCCAC
3G5 Rev
AAA CACGATAA CAC
TGGGGG
6G2 Rev
CCCTTTGCTTACCC
AAG TTGCT
5G4 For
AGTAGGCAGA TAA
GGGGTTTCC
MusY For
TCCTTGGGCTCTTC
ATTATT CTTAAC
5G4 Rev
TACAGCATCTAGTG
AAT GGGGG
MusY Rev
GAGAAC CACGTTG
GTTTGAGAT G
What is a DNA polymorphism?
How are you going to use DNA
polymorphisms in this
experiment?
Example from previous Year
5G4
1
2
3
4
5
6
7
2
3
4
5
6
7
6G2
1
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 Electrophoresis
To 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
30% acrylamide/bis
5X TBE
10% APS
temed
water
total
Volume
4ml
3ml
45l
15l
7.9
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 snacking
Larger portions
Eating 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
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7