in vitro repair enhances amplicon recovery and accuracy from damaged

DNA

Tom Evans, Ph.D.

New England Biolabs, Inc.

Accessing Genetic Informationpost-mortem

• Variables to Genetic Quality.– Storage state.

• Purified.• in situ.

– Post-mortem interval.– Storage environment.

• Frozen.• Dried.• Ethanol.• Formalin treated.

Accessing Genetic Informationpost-mortem

• Limiting Factor.– DNA extraction efficiency.– PCR/Sequencing inhibitor co-purification.– DNA quality.

Solutions

• Mitochondrial Barcode.– Copy number.

• Minimal Barcode sequence.

• Superior DNA extraction protocols.

• Improve DNA quality.

• More robust analytic techniques/enzymes.

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.

DNA DamageMutagenic Lesions

• Deaminated Cytosine.– C to T transition.

• Oxidative Lesions.– 8-oxo-guanine.

• G to T transversion.

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.

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

PreCRRepair Spectrum

Repair Spectrum

Abasic sites

Nicks

Gaps

Blocked 3’ Ends

Oxidized Purines

Oxidized Pyrimidines

Thymine Dimers

Deaminated Cytosine

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.

PreCRRepair Spectrum

DOES NOT REPAIR

Double Strand Breaks

DNA-Protein Cross-links

DNA-DNA Cross-links

PreCRUV Damaged DNA

DNA (ng):

3 min UV 4 min UV 5 min UV 10 min UV

5 2.5 1

0.5 5 2.5

1 0.5

5 2.5 1 0.5

5 2.5

1 0.5

5 2.5 1 0.5

5 2.5

1 0.5 5 2.5 1 0.5

5 2.5

1 0.5

PreCR: - + - + - + - +

Oxidized DNA

Clone Amplicon into Expression Plasmid.

Transform intoE. coli.Grow on X-GalPlates.Expose DNA template to light

in the presence of methylene blue.

Use repaired or unrepairedtemplate in PCR.

thermocycler Sequence

PreCROxidatively Damaged DNA

TABLE 1. Base substitution errors after PCR amplification of plasmid DNA (4766 bp amplicon), untreated, treated with methylene blue (MB), and repaired with two different repair mixes at 37oC for 15 min. Substitutionsa Control, no

MB 1)c 25 M MB

4) 50 M MB 2) PreCR-A 25 M MB

5) PreCR-A 50 M MB

3) T21 25 M MB

6) T21 50 M MB

Total bp sequenced

30,128 30,128 N/A 15,064 15,064 15,064 15,064

Per 103 bp 0.03 1.4 N/A 0.13 0.0 0.0 0.0 25 g/µL Methylene Blue 50 g/µL Methylene Blue no repair PreCR-A T21 no repair PreCR-A T21 1) 2) 3) 4) 5) 6)

Oxidatively Damaged DNA Even Worse Damage

PreCR PreCR

0

20

40

60

80

0 20 40 60 80 100 120 140 160

# o

f A

P s

ite

s/1

05 b

p

Citrate Incubation Time (min)

PreCR Activity on AP Sites

pH 5 Incubation Time (min)

PreCR treatment

No treatment

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– PreCR allows access (amplification) to

more heavily damaged templates than was possible previously.

Extent of DNA damage

PC

R y

ield maximal

failed0 high

PreCR treateduntreated

Real World Issues

• What is the most common limitation?– DNA extraction.– PCR inhibitors.– DNA Quality.

• Base damage.• Backbone breaks.

Acknowledgements

Barton Slatko Romas Vaisvila Lixin Chen Peter HartlineElizabeth Cantin Katherine MarksDakota 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.