Régis DEBRUYNE Auxiliaire de recherche ADNs dégradés & NTS Service de Systématique Moléculaire, UMS 2700, Dépt.

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1 Régis DEBRUYNE Auxiliaire de recherche ADNs dégradés & NTS Service de Systématique Moléculaire, UMS 2700, Dépt. SE

2 All biological macromolecules spontaneously decompose. So does DNA This degradation is not a pure stochastic process; It can be modelized pretty accurately 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 2

3 All biological macromolecules spontaneously decompose. So does DNA In metabolically active cells, DNA repair mechanisms counter the effect of damage Rich enzymatic machinery: abasic sites removal single base-pair excision mismatch repair single strand break repair double strand break repair 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 3

4 All biological macromolecules spontaneously decompose. So does DNA ANCIENT DNA THE ORIGINAL 4 FORENSICS In metabolically active cells, DNA repair mechanisms counter the effect of damage DNA damage accumulates in metabolically impaired or inactive cells Various situations involved: organismal death cellular death loss of organismal integrity change in cell behaviour change in chemical environment ONCOLOGY MUTAGENESIS 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 4

5 NATURAL HISTORY MUSEUMS All biological macromolecules spontaneously decompose. So does DNA ANCIENT DNA In metabolically active cells, DNA repair mechanisms counter the effect of damage DNA damage accumulates in metabolically impaired or inactive cells Various situations involved: organismal death cellular death loss of organismal integrity change in cell behaviour change in chemical environment 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 5

6 NATURAL HISTORY MUSEUMS All biological macromolecules spontaneously decompose. So does DNA ANCIENT DNA COLL. DNA In metabolically active cells, DNA repair mechanisms counter the effect of damage DNA damage accumulates in metabolically impaired or inactive cells Various situations involved: organismal death cellular death loss of organismal integrity change in cell behaviour change in chemical environment ENV. DNA NON-INVASIVE SAMPLING 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 6

7 Time is not the only key 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 7

8 Free water effect of ph Reactive oxygen peroxides hydroxyls Energy Heat Light (UV) ionizing radiation Chemicals self biomolecules foreign molecules cumulative effects through time Hydrolysis Oxidation Helix distortion Condensation ( crosslinks ) 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 8

9 2000 / AP sites per cell per day glycosidic bond DNA RNA phosphodiester bond depurination rate = 20x depyrimidation rate 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 9

10 Cytosine deamination 06 Novembre 2012 T 5mC non 5mC ADN dégradés et ADN anciens au MNHN 10 U

11 Polymerase non-bypassable damage 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 11

12 Partly methylated nuc genome low oxygen content protein quenching ±180 bp units Non-methylated mt genome oxygen metabolism no protein association 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 12

13 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 13

14 MODERN SAMPLES For live specimens Collection initiates DNA degradation >> post-mortem degradation starts immediately after sampling For non-invasive and environmental samples >> Low concentration and non-cellular state drastically reduce the survival time of DNA ANCIENT SAMPLES Collection potentially increases damage by the disruption of the environmental conditions >> exposure to light, oxygen and humidity of buried specimens... via rapid and/or adequate sampling 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 14

15 Total DNA count comparison: 10x more than 5-year room-temp. preserved specimens x more than a 100-year old collection yet it is seldom possible 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 15

16 Molecular sampling usually takes place way after actual on-site collection. Time in preservatives or dried states : rapid decay of DNA Strong concentration of chaotropic salt + silica-based extraction hydrophobic environment protein denaturation DNA immobilization 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 16

17 Forensic device: FTA cards Lightweight Non-hazardous FTA dry matrix: Silica fibers Guanidinium salt 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 17

18 water maceration (+ additives) detergent enzyme mix heat oxidizing agents (bleach etc.) alkaline treatment organic solvent degreasing burial (soil contamination) dermestid beetles digestion not compatible with metagenomic studies 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 18

19 Formalin is a formaldehyde-saturated (~30%) water buffer Reversible crosslinks Formalin fixation (buffered/non-buffered) 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 19

20 DNA-DNA Benefit: Effectively preserve structural morphology Drawbacks: Stall polymerase Prevent denaturation Cause co-purification of DNA and protein using organic solvent Type of crosslinks DNA-protein 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 20

21 Formalin is a formaldehyde-saturated (~30%) water buffer Reversible crosslinks Formalin fixation (buffered/non-buffered) Paraffin embedding Irreversible crosslinks Accelerated hydrolysis Formalin preserving Oxidation to formic acid with time Ethanol preserving Cell membranes disruption > DNA release 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 21

22 Deplete total amplifiable DNA Pre-extraction incubation at high T Long digestion time Reverse crosslinks Remove inhibitors Denature proteins Break Maillard compounds Hot alcali treatment 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 22

23 Serial quantitative assay (12S for vertebrates) Real-time amplification profile 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 23

24 Copy number calculation 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 24

25 Log transformed copy N 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 25

26 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 26

27 B form A form Benefit: DNA dessiccation allows long-term preservation at room-temperature Drawback: Limited availability of the DNA for molecular use Unsolved question: Induced fragmentation by removal of structural water molecules from DNA grooves? 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 27

28 endogenous DNA contaminant DNAs 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 28

29 Old-fashioned targeted PCR Huge waste of endogenous DNA Rapid use of extracts requires the repeated sampling of the specimen 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 29

30 Whole bank replication (no re-sampling of specimens) Benefit: DNA readily available for analysis Drawbacks: Doesn t account for the freezer contraint Risk of molecular bias during whole bank replication 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 30

31 < 1% mtdna > 99% nucdna Genomic copy number (via targeted PCR) Until 2005, less than 2% of adna studies aimed at nucdna 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 31

32 < 1% < 1% > 99% mtdna nucdna > 99% Genomic copy number (via targeted PCR) Actual DNA amount in extracts (via shotgun sequencing) 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 32

33 BAC-clones genomics 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 33

34 Degraded DNA Are theoretically well-adjusted To NGS sequencing needs BAC-clones genomics NGS-based genomics 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 34

35 < 5% neand. BAC-clones genomics NGS-based genomics > 95% other non-permafrost neandertal remains 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 35

36 Biotinylated specific probes 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 36

37 Biotinylated specific probes Specific recovery of endogenous DNA via magnetic separation 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 37

38 06 Novembre 2012 ADN dégradés et ADN anciens au MNHN 38