engelward, spring 2008 - amazon s3 · 2017-01-26 · engelward, spring 2008 day 3. ... primer and...
TRANSCRIPT
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MOD1 DNA ENGINEERINGMOD1 – DNA ENGINEERING
Engelward, Spring 2008
D 3Day 3
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About the experiments in Mod1 ‐how is recombination used to fix double strand breaks‐how your two‐plasmid assay works
A G l H d ‘l k’ t DNA?Agarose Gels – How do we ‘look’ at DNA?
Anticipating Potential Problems & PitfallsAnticipating Potential Problems & Pitfalls
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Background & Significance:Background & Significance:
“Homology‐Directed Repair” for double strand breaksstrand breaks
You will need to understand this material in order to write your final report.
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Why you owe Your Life to Homologous Recombination…
Normal Rad51‐/‐
Turn Off Homologous Recombination → Chromosomes Fall Apart→ Chromosomes Fall Apart
Sonada et al., EMBO J. 17, 598–608 (1998).
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Why you owe Your Youthfulness to Homologous RecombinationHomologous Recombination…
Loss of Helicase→ Faulty RecombLoss of Helicase → Faulty Recomb.
’Werner’s Syndrome
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Double Strand Breaks
NHEJ HR
KU heterodimer (Ku70&80)DNA‐PKcsDNA ligase IV XRCC4
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See NHEJ Animation by Justin LoSee NHEJ Animation by Justin Lo
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Double Strand Breaks
NHEJ HR
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=
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Imagine HR is initiated by the fragment on the left….
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Step 1: A double stranded end has been created
Step 2: Resect the end toStep 2: Resect the end to create a 3’ overhang
Step 3: Create a nucleoprotein filament capable of homology searching
Egelman, PNAS
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Step 4: Search and Invade
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Step 4: Search and Invade
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Step 4: Search and Invade
Step 5: Polymerize DNA using invading strand with 3’OH as a primer and the homologous donor DNA as a template
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Step 6: Branch Migration (Backwards)Step 6: Branch Migration (Backwards)
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Step 6: Branch Migration (Forwards)Step 6: Branch Migration (Forwards)
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Step 6: Possible ReleaseStep 6: Possible Release
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This process started with a two‐ended DSB...
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Now let’s imagine the same thing happened at the other end…the other end…
Annealing
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Final Steps: Filling Trimming LigatingFinal Steps: Filling, Trimming, Ligating
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++
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See SDSA AnimationSee SDSA Animationby Justin Lo
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E. coli: RecA*
Yeast: Rad51Yeast: Rad51
Mammals: RAD51
Egelman, PNAS 200
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See Prototypic Model AnimationSee Prototypic Model Animationby Justin Lo
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DNA Damage can be repaired by Homology Directed Repair (HDR)
This is the ‘prototypic’ model of repair of how homologous recombination can repair a
double strand break
NOTE: BREAKPOINTNOTE: BREAKPOINT TURNS FROM BLUE TO
RED
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Decision to initiate HDR,resection of DNA ends
ATM, ATR, cAbl, Chk1, p53, BRCA1, Fanconi genes, Mre11, Rad50, Nbs1, Exonucleasesresection of DNA ends
Displacement of RPA &Loading Rad51
RPA, Rad52, BRCA2,Rad51, Rad51B, Rad51C, gRad51D, XRCC2, XRCC3
Homology searching, histone remodeling, & invasion
Rad54, Rad54B, Rad52
MMR, WRN, BLM, Rad54, p53Holliday junction migration, inhibition by mismatches
Repair synthesis, Holliday junction migration, Polymerase(s), topoisomerase(s)
WRN BLM Rad54Possible resolution without junction cleavage
Rad51C & possible additional
WRN, BLM, Rad54
Junction resolutionRepair of mismatches
Rad51C & possible additional proteins; possible resolution by topoisomerases; MMR
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Agarose Gels & Gel PurificationAgarose Gels & Gel Purification
How do we ‘look’ at DNA?‐ How do we ‘look’ at DNA?‐ How do we get our DNA out of a gel?
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How can you see your DNA?
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Gel Purification
Why do you need to cut out your band y y yfairly quickly?
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Sunlight Damages DNA
N NH2 O
NN HNA TN
N O H2N
N
O
NHNN
H2N
G CN
NH2
N
O
C
Normal Base Pairs
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Sunlight Damages DNA
N NH2 O
NN HNA TN
N O H2N
N
O
NHNN
H2N
G CN
NH2
N
O
C
Sunlight‐Induces Crosslinks Between Bases
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Sunlight Damages DNA
Before After
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Gel Purification
Why do you need to cut out your band y y yfairly quickly?
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You will need to dissolve the gel to get the DNA out.. You do this by adding 3 volumes of y g
a gel‐dissolving solution.
What does it mean to ‘add 3 volumes’?
How can you estimate the volume of your gel slice?y g
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Agarose Gels – How do we ‘look’ at DNA?Agarose Gels – How do we look at DNA?
‐Loading
‐Standards
‐Parameters that affect migrationParameters that affect migration
‐gel concentration‐length of DNA‐tertiary structure‐effects of overloading
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Overview of theOverview of the Experiments in Mod1
Where you areWhere you are,and where you are going
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A Plasmid-Based Assay for Homologous Recombination in Mammalian Cells
Δ5Δ5
+lipofect 48 hours
Δ3
+
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X EPCR1
Purif. X E
2Digest X E
2 GelPurif. X E
3
EX
EX EX3GelAnal.
3
3 X E 4 4PlanLig.
3 X E
Ligate4
E. coliE. coli
E. coliE. coli
Transform4
5
Purif5
Digest GelAnal.
6
Transfect7
Flow
8
Purif.DNA
Flow
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X EPCR1
Purif. X E
2Digest X E
2
EX
EX
How do you know that your restriction enzymes actually cutenzymes actually cut
the DNA?
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X EPCR1
Purif. X E
2Digest X E
2
EX
EX
What else is in the reaction with the digested PCR product?
What effect could it have?
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X EPCR1
Purif. X E
2Digest X E
2
EX
EX
GelPurifPurif.
Why is it important to excise the DNA y pfrom the gel relatively quickly?
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X EPCR1
Purif. X E
2Digest X E
2 GelPurif. X E
3
EX
EX EX3GelAnal.
3
Why run this gel?
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X EPCR1
Purif. X E
2Digest X E
2 GelPurif. X E
3
EX
EX EX3GelAnal.
3
Your objective is a 1:4 vector:insert ratio – Why?y
What if it was 1:100?What if it was 1:100? What if it was 100:1?
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X EPCR1
Purif. X E
2Digest X E
2 GelPurif. X E
3
EX
EX EX3GelAnal.
3
How do you figure out how to get a 1:4 molar ratio?
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About the experiments in Mod1 ‐how is recombination used to fix double strand breaks‐how your two‐plasmid assay works‐overview of the experiments you will be doing
A G l H d ‘l k’ t DNA?Agarose Gels – How do we ‘look’ at DNA?‐what is a gel and how do you load it?‐what happens to your DNA when it is exposed to UV?‐parameters that affect migration
Anticipating Potential Problems & Pitfalls‐Getting the right DNA ratios