Transcript PreCR DNA Repair Mix - Smithsonian Institution

in vitro repair enhances amplicon
recovery and accuracy from damaged
DNA
Tom Evans, Ph.D.
New England Biolabs, Inc.
Accessing Genetic Information
post-mortem
• Variables to Genetic Quality.
– Storage state.
• Purified.
• in situ.
– Post-mortem interval.
– Storage environment.
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Frozen.
Dried.
Ethanol.
Formalin treated.
Accessing Genetic Information
post-mortem
• Limiting Factor.
– DNA extraction efficiency.
– PCR/Sequencing inhibitor co-purification.
– DNA quality.
Solutions
• Mitochondrial Barcode.
– Copy number.
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Minimal Barcode sequence.
Superior DNA extraction protocols.
Improve DNA quality.
More robust analytic techniques/enzymes.
Improve DNA Quality
Damages that Inhibit Primer Extension
• Depurination/Depyrimidation.
– Most common damage under physiological conditions.
• Oxidative Lesions.
– Thymine glycol.
– Certain species of oxidized guanine?
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Thymine Dimers.
Nicks.
Double Strand Breaks.
DNA-protein and DNA-DNA cross-links.
DNA Damage
Mutagenic Lesions
• Deaminated Cytosine.
– C to T transition.
• Oxidative Lesions.
– 8-oxo-guanine.
• G to T transversion.
Improve DNA Quality
Goals
• Full repair in one-pot without sequential enzyme
addition, enzyme inactivation, or DNA purification.
• Easy reaction optimization.
• Does not hurt the reaction, even if it does not
help.
• Full repair.
• Not tied to one protocol.
How does it work?
Damage Recognition
Endonuclease IV
How does it work?
Nick translation
Polymerase (5’-3’ exo+)
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Nick translation
How does it work?
Polymerase dissociation
How does it work?
Nick ligation
Taq DNA ligase
How does it work?
Nick ligation
How does it work?
Nick ligation
How does it work?
Nick ligation
PreCR
Repair Spectrum
Repair Spectrum
Abasic sites
Nicks
Gaps
Blocked 3’ Ends
Oxidized Purines
Oxidized Pyrimidines
Thymine Dimers
Deaminated Cytosine
PreCR
Enzyme Composition
It’s now 7 enzymes.
Taq DNA ligase.
E. coli endonuclease IV.
Bst DNA polymerase I.
E. coli Fpg.
E. coli Udg.
T4 pdg.
E. coli endoVIII.
PreCR
Repair Spectrum
DOES NOT REPAIR
Double Strand Breaks
DNA-Protein Cross-links
DNA-DNA Cross-links
PreCR
UV Damaged DNA
PreCR:
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+
3 min UV
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4 min UV
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5 min UV
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10 min UV
Oxidized DNA
Clone Amplicon
into Expression
Plasmid.
Expose DNA template to light
in the presence of methylene
blue.
Transform into
E. coli.
Grow on X-Gal
Plates.
Use repaired or unrepaired
template in PCR.
thermocycler
Sequence
PreCR
Oxidatively Damaged DNA
Oxidatively Damaged DNA
PreCR
Even Worse Damage
PreCR
PreCR Activity on AP Sites
5
# of AP sites/10 bp
80
No treatment
60
40
20
PreCR treatment
0
0
20
40
60
80
100
120
Citrate
Incubation
Time(min)
(min)
pH
5 Incubation
Time
140
160
Real World Issues
• Unknown damage.
– There are few studies on what is actually wrong with stored
DNA.
• Jürgen Zimmermann is characterizing DNA damage in moth
samples, see poster. Poster abstract on page 154.
• Unknown DNA.
– Unknown DNA quantities.
• PCR inhibitors.
– BSA tube in PreCR Repair Mix helps deal with PCR
inhibitors.
PreCR
• Perception
PCR yield
– PreCR allows access (amplification) to
more heavily damaged templates than was
possible previously.
maximal
PreCR treated
untreated
failed
0
high
Extent of DNA damage
Real World Issues
• What is the most common limitation?
– DNA extraction.
– PCR inhibitors.
– DNA Quality.
• Base damage.
• Backbone breaks.
Acknowledgements
Barton Slatko
Vaisvila
Lixin Chen
Elizabeth Cantin
Dakota Hamill
Romas
Peter Hartline
Katherine Marks
Mehrdad Hajibabei, University of Guelph.
Lee Weigt, NMNH-LAB.
Ann Bucklin, University of Connecticut.
James Hanken, MCZ, Harvard University.
David Blackburn, MCZ, Harvard University.
David Schindel, CBOL Executive Secretary.
Christoffer Schander, University of Bergen.
Jan E. Janecka, Texas A&M University.
John V. Planz, UNTHSC.