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in vitro repair enhances amplicon recovery and accuracy from damaged DNA

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. Frozen. Dried.

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in vitro repair enhances amplicon recovery and accuracy from damaged DNA

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  1. in vitro repair enhances amplicon recovery and accuracy from damaged DNA Tom Evans, Ph.D. New England Biolabs, Inc.

  2. Accessing Genetic Informationpost-mortem • Variables to Genetic Quality. • Storage state. • Purified. • in situ. • Post-mortem interval. • Storage environment. • Frozen. • Dried. • Ethanol. • Formalin treated.

  3. Accessing Genetic Informationpost-mortem • Limiting Factor. • DNA extraction efficiency. • PCR/Sequencing inhibitor co-purification. • DNA quality.

  4. Solutions • Mitochondrial Barcode. • Copy number. • Minimal Barcode sequence. • Superior DNA extraction protocols. • Improve DNA quality. • More robust analytic techniques/enzymes.

  5. Improve DNA QualityDamages that Inhibit Primer Extension • Depurination/Depyrimidation. • Most common damage under physiological conditions. • Oxidative Lesions. • Thymine glycol. • Certain species of oxidized guanine? • Thymine Dimers. • Nicks. • Double Strand Breaks. • DNA-protein and DNA-DNA cross-links.

  6. DNA DamageMutagenic Lesions • Deaminated Cytosine. • C to T transition. • Oxidative Lesions. • 8-oxo-guanine. • G to T transversion.

  7. Improve DNA QualityGoals • 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.

  8. How does it work?Damage Recognition Endonuclease IV

  9. How does it work?Nick translation Polymerase (5’-3’ exo+)

  10. How does it work?Nick translation

  11. How does it work?Nick translation

  12. How does it work?Nick translation

  13. How does it work?Nick translation

  14. How does it work?Nick translation

  15. How does it work?Nick translation

  16. How does it work?Nick translation

  17. How does it work?Nick translation

  18. How does it work?Nick translation

  19. How does it work?Polymerase dissociation

  20. How does it work?Nick ligation Taq DNA ligase

  21. How does it work?Nick ligation

  22. How does it work?Nick ligation

  23. How does it work?Nick ligation

  24. PreCRRepair Spectrum

  25. PreCREnzyme 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.

  26. PreCRRepair Spectrum

  27. PreCRUV Damaged DNA PreCR: - + - + - + - + 2.5 0.5 2.5 2.5 2.5 2.5 1 1 2.5 2.5 DNA (ng): 2.5 0.5 1 1 5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 5 5 5 5 1 1 5 5 5 3 min UV 4 min UV 5 min UV 10 min UV

  28. Oxidized DNA Clone Amplicon into Expression Plasmid. Transform into E. coli. Grow on X-Gal Plates. Expose DNA template to light in the presence of methylene blue. Use repaired or unrepaired template in PCR. Sequence thermocycler

  29. PreCROxidatively Damaged DNA Oxidatively Damaged DNA Even Worse Damage PreCR PreCR

  30. PreCR Activity on AP Sites No treatment PreCR treatment pH 5 Incubation Time (min)

  31. 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.

  32. PreCR • Perception • PreCR allows access (amplification) to more heavily damaged templates than was possible previously. maximal PreCR treated PCR yield untreated failed 0 high Extent of DNA damage

  33. Real World Issues • What is the most common limitation? • DNA extraction. • PCR inhibitors. • DNA Quality. • Base damage. • Backbone breaks.

  34. Acknowledgements Barton SlatkoRomas Vaisvila Lixin ChenPeter Hartline Elizabeth Cantin Katherine Marks Dakota Hamill 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.

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