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

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Presentation on theme: "In vitro repair enhances amplicon recovery and accuracy from damaged DNA Tom Evans, Ph.D. New England Biolabs, Inc."— Presentation transcript:

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

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

3 Accessing Genetic Information post-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 Quality Damages 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 Damage Mutagenic Lesions Deaminated Cytosine. –C to T transition. Oxidative Lesions. –8-oxo-guanine. G to T transversion.

7 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.

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

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19 How does it work? Polymerase dissociation

20 How does it work? Nick ligation Taq DNA ligase

21 How does it work? Nick ligation

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24 PreCR Repair Spectrum Repair Spectrum Abasic sites Nicks Gaps Blocked 3’ Ends Oxidized Purines Oxidized Pyrimidines Thymine Dimers Deaminated Cytosine

25 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.

26 PreCR Repair Spectrum DOES NOT REPAIR Double Strand Breaks DNA-Protein Cross-links DNA-DNA Cross-links

27 PreCR UV Damaged DNA DNA (ng): 3 min UV4 min UV 5 min UV 10 min UV PreCR:

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. thermocycler Sequence

29 PreCR Oxidatively Damaged DNA Oxidatively Damaged DNAEven Worse Damage PreCR

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

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. Extent of DNA damage PCR yield maximal failed 0high PreCR treated untreated

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

34 Acknowledgements Barton Slatko Romas Vaisvila Lixin ChenPeter Hartline Elizabeth CantinKatherine 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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