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Molecular genetic investigations in natural history collections

--- Introductory seminar on the use of molecular too ls in natural history collections - 6-7 November 200 7, RMCA ---

From specimens to DNA•Specimen, tissue and DNA extract collections•DNA damages in archival specimens•Preservation issues (specimen, tissue and DNA storage)

DNA extractions from museum specimens•DNA extraction methods•Precaution and controls •DNA extraction from fluid preserved collections

•Alcohol stored and formalin fixed specimens•DNA extraction from dry collections

•Whole animals, plant collections, skins, hard tissues, (sub)fossils, etc.


From specimens to DNA

Museum curationSpecimen banks (voucher numbers)Data banksType specimens

Molecular workTissue banks, DNA extract banksGene banks (sequence identifier)

•Barcodes•Others DNA sequences

Voucher specimen >Tissue sample >DNA extract >PCR product >DNA Sequence


Genetic sequence database: GenBankPublic gene banks:GenBank (USA)EMBL (Europe)DDBJ (Japan)

Entries:•Accession number•Locus•Organism•Reference (author, title, journal, etc.)•Etc.

Keyword or BLAST searches (Basic Local Alignment Search Tool)http://www.ncbi.nlm.nih.gov/Genbank/index.html

Annotated collection of all publicly available DNA sequences.

>60 109 bases in >60 106 sequence records in the traditional GenBankdivisions


From specimens to DNA

< Rowley et al. 2007


DNA damages in archival specimens

After the death of the organism:•No more reparations of the DNA•Catabolic enzymes are no more sequestrated (e.g. lysosomalnucleases)•Development of parasites (bacteria, fungi, insects)

In living cells:•Molecule maintenance•Preservation of cellular compartments

•Defense of the organisms against parasites

Effect of conservation and fixative agents•Conditions (T°, pH, humidity%, etc.) •Preservation treatments (formalin, ethyl acetate, etc.)



� Enzymatic degradationnucleases in post mortem cellsdegradation by microorganisms

� Non enzymatic degradationoxidative lesionshydrolytic lesions

� Radiations

•Double strand breaks •Single strand breaks•Sugar fragmentation•Base modifications•Crosslinks



< Austin 1997



A deamination example :

Cytosine � Uracil

Preservation issues- Storing specimens and samples for use in

molecular genetic analysis -


Aim of storage:•Long-term storage•Quality: mtDNA, nuclear DNA, RNA or protein analyses•Avoiding contaminations•Non-destructive sampling (e.g. feather, hair, exuvium, feces)

Feasibility:•Volume of samples•Budget allowance•Time frame•Preparation•Field work

Preservation issues- Storing specimens and samples for use in

molecular genetic analysis -


•Chemical desiccation : hexamethyldisilazane (HMDS); amyl acetate; xylene

•Physical desiccation : sun/air drying; silica gel/beads; NaCl; CaSO4 (dierite)

•Storage in fluids: ethanol; other alcohols; propylene-glycol; glycerol; DMSO/salt; lysis buffer; acetone; thymol buffer; trehalose.

•Freezing: -20; -80; liquid nitrogen storage

•Special kits: RNALater ; FTA paper Guthrie cards; GT-903

Diluted DNA storage


•TE buffer (Tris pH buffer and EDTA, Ethylene diamine tetracetic acid)

•Lambda DNA (Hind III digested lambda-DNA)

•Trehalose(Disaccharide of glucose)

•Storage in water is subjected to acid hydrolysis

< Smith et al. 2005

DNA extraction from museum specimens- Important steps -


The tissue preparation•Choosing the specimens (sp., age, geographic origin, previous studies, etc.)•Tissue sampling (tissue, amount of starting material, control, etc.)•Decontamination•Fragmentation, perforation, homogenization•Tissue lysis (mechanical and chemical treatments)(SDS, CTAB, DTT, prot.K, heating)

Nucleic acid isolation and purification•Choosing the method•Further adaptations for museum specimens

Nucleic acid quality evaluation•Spectrophotometry•Electrophoresis on agarose gel

Different methods for DNA extraction:

• Organic extraction (e.g. Phenol/PCI)

• Denaturation and dilution (e.g. Chelex)

• Differential precipitation (e.g. salting out the DNA)

• Differential affinity (e.g. silica gel)

• Differential affinity (e.g. magnetic beads)


DNA extraction methods

• e.g. Phenol/Chloroform :

+ Universal method for purification of nucleic acids

Good yield

- Time consuming

Toxic


DNA extraction methods- Organic extraction -

Aqueous phase

Interphase

Organic phase

Although each of these solvents is capable of performing this function alone, the two materials together remove proteins from solution much more effectively. Nucleic acids are recovered in the liquid phase.

Proteins are removed by phenol and chloroform in disrupting protein secondary structure causing proteins to denature and precipitate from solution

DNA extraction methods- Denaturation -

• e.g. Chelex:

+ less manipulations� faster and less contamination riskscheap

- not universalless yield


Solution is transferred to crushed ice and then centrifuged. The chelex precipitates along with the denatured protein. The DNA is in the supernatant

The cells are lysed and proteins denatured by exposing to 100 °C for ten minutes.

Chelex resins are weak cationchelating exchangers. It removes polyvalent metal ions which might enhance DNA breakdown during boiling or inhibit PCR reactions.

• e.g. salting out the DNA:

+ Non toxicCheap

- Not universal


DNA extraction methods- Differential precipitation -

This method involves salting out of the cellular proteins by

dehydration and precipitation with a saturated NaCl solution.

• e.g. spin columns with silica membrane :

+ Easy and fastClean product for PCR

- Lower yield


DNA extraction methods- Differential affinity -

DNA is absorbed onto a silica-gel membrane during a first centrifugation. Salt and pH conditions in the lysate �

Proteins and other contaminants (that can inhibit PCR reaction are not

retained

2 washing steps to remove residual contaminants

DNA is retained on membrane

Tissue is lysed 2 Times elution


DNA extraction methods- Differential affinity -

• e.g. magnetic beads:

+ Easy and fastClean product for PCR

- Lower yieldNot universal

DNA is captured on magnetic beads leaving most of the cell debris and proteins in solution. Then any residual protein is digested and washed away. Pure DNA

is finally eluted.


DNA extraction methods- Adaptations -

Improvement of yield or concentration:•2 times elution step •Incubation at room temperature•Effect of elution volume:


Evaluation of DNA extracts- Spectrophotometry analysis -

Information given by the NanoDrop :

•DNA concentration (in ng/µl)

•260/280 ratio

•260/230 ratio

•Etc.


Evaluation of DNA extracts- Spectrophotometry analysis -

Effect of UV exposition:


Evaluation of DNA extracts- Total DNA electrophoresis -

Electrophoresis on agarose gel 1,2%

Separation of theDNA moleculesaccording to theirsize

28 10 2 38 9 137 49 32712 7 192 ngDNA/µl

2? 24 76 2 8 270 38 32 339 Smart ladder

DN

A m

igra

tion

DN

A m

igra

tion

Anod +

Cathod -


1965 1966 1973 1977 1979 1980


Effect of specimen’s age:

DNA extracted from birds footpads collected at different

dates

Date of collection:


After 2 years of storage:

DMSO-salt sol

lysis buffer

T. stored in lysis buffer

T. stored in ethanol + lysis buffer

stored in ethanol


Effect oftissue storageconditions:

(DNA extractions

after 2 years ofDNA storage)

<Kilpatrick 2002



Effect ofkilling andsoragemethod:

< Dean & Ballard 2001

Joint Experimental Molecular UnitJEMU



1. Silica gel, 2 years2. Silica gel, 2 month, 3. 2-propanol4. In Carnoy’s solution5. 75% ethanol at RT6. 75% ethanol at 4°C7. 100% ethanol at RT8. 100% ethanol at 4°C, 1 month9. 100% ethanol at 4°C, 1 year10. 100% ethanol at 4°C, 2 years11. Acetone, 2 years12. In an ultracold freezer 13. In liquid nitrogen14. Freshly captured15. Freshly captured after desiccation

Freshly collected and stored into EtOH 80% (Sufficiently high to preserve DNA but not so high as to cause desiccation and distort the animal’s external morphology.

< Mandrioli et al. 2006



DNA extraction from museum specimens- DNA quality �� PCR success rate -

Pääbo et al. 2004

< Latepleistocene



DNA extraction from museum specimens- Precautions -

The risk of contamination is high:

� Low DNA concentration� Manipulations by humans� Parasites � Contact with other specimens

�Did we sequence the right DNA ? Carpet beetleAnthrenus

verbasci

e.g. a cave bear tooth was found to contain 20 different humanSequences !



DNA extraction from museum specimens- Precautions -

•Sterile area and sterile instruments at all stages (gloves, filter pipette tips)

•Separation of the labs: •Pre- PCR >< post- PCR labs•Ancient DNA lab >< modern specimens lab

•Extraction blanks and PCR blanks.

•Random extraction from different groups and blind testing

•Multiple DNA extraction for each specimen

•Multiple PCR of each locus for each specimen

•Cloning of PCR products and DNA sequence analysis

•Appropriate results (sequence similarity between close relative specimens)

•Reproduction in a second laboratory



DNA extraction from museum specimens- Controls -

If amplification:�Right DNA fragment�Widespread contamination ???NEGATIVE CONTROL

If no amplification:�Degraded DNA�Something wrong in the PCR ???POSITIVE CONTROL

Or

�PCR inhibitors in sampleINTERNAL CONTROL ???(positive DNA control in suspected sample)



DNA extraction from museum specimens- Fluid preserved collections -

•Alcohol collections

•Ethanol 70 – 100%

•Isopropanol

•Methanol

•Acetic alcohol collections (Carnoy’s solution, ethanol-chloroform-acetic acid70:20:10)

•Formalin fixed specimens

Natural history collection - The University of Kansas



DNA extraction from museum specimens- Alcohol collections -

Conservation: < 200 days at 4°C in EtOH 80%

Whole specimens fully immersed in digestion buffer and incubated overnight.

Phenol:chloroform extraction followedby isopropanol precipitation.

Specimens were removed from the buffer, placed in 100%EtOH for 2–4 hours to stop further digestion, air-dried, and replaced intheir collections.

Raioella indica

< Rowley et al. 2007




UnextractedControlUnextracted

Control

extracted for 2 h and ethyl acetate-dried

Wing extracted for 4 h

Wing extracted for 1 h

extracted for 2 h and

critical-point-dried

Delphinia picta <Rowley et al. 2007




Until now, bad results with plant collections

� Dried plant collections



DNA extraction from museum specimens- Formalin fixed specimens -

DNA alterations:

•Fragmentation•Sequence modification•Cross-linking

Rapid reactions between DNA and formaldehyde (generally reversible) +Further reactions over the long term (not well characterized)




Variation in curatorial practices for specimen storage:

•How long in formalin ?•Long-term storage in alcohol or in formalin ?•10-37% formaldehyde ? pH 7 ? buffered or unbuffered ?

� no optimal protocol

10% buffered Form.10% buffered Form.>1960

70% ETOH10% Formalin1890-1960

ETOH/SpiritsETOH/Spirits<1890

PreservativeFixativeYears

History of the Amphibian larval fixation and

preservation traditions at the United States National

Museum (USNM )




Reactions of formaldehyde with nucleotides and nucleic acids (under 24h):

Could be reversed by incubation at 70°C or dialysis

BUT - Fixation at 24°C- Not tranferred to ethanol




If formaldehyde is unbuffered, it is oxydized to form formic acid. •Acidification causes depurination resulting in the loss of purine from the DNA backbone.

Formation of oxidative adducts that lead to mutagenic lesions: •Cytosine deamination (could be enzymatically reversible)•Modifications to adenosine•Etc.

Depurination: cleavage of the N glycosidic link between purine bases and deoxyribose in DNA. Degradation is likely to be irreversible




�Better selection of the samples and the DNA extraction methods �Knowledge on the treatments of the formalin fixed samples is very important !!!

•DNA from some tissues are more resistant to formalin alteration (teeth and bones)•Samples kept in unbuffered formalin are not likely to be usable because of formic acid damage, •Samples with free purines could be detected by mass spectrometry

<Tang 2006




e.g. Small specimens: Nematodes or deep-sea mollusks

History: fixed in formalin and mounted in anhydrous glycerol onto glass microscope slides < 30 years

Protocol: hot lysis protocol (higher volume of buffer)Nematodes were picked out from microscope slides

Fragments of 500-600 base pairs were the longest that had been obtained from formalin-fixed samples stored in aqueous solution.

< Bhadury et al. 2007



DNA extraction from museum specimens- Dry collections -

•Whole animal

•Plant collection

•Skin, scale, feather and other (exuvia,dried snakevenom)

•Hard tissue (bone, skull, shell)

•(Sub)fossil



DNA extraction from museum specimens- Arthropods -

Material depending on the size:

•Whole specimens •Perforated by small insect pins•Fully immersed in digestion buffer

•Leg (e.g. tibia) �1870 BUT 73bp•Crushed and soaked in ethanol for a few minutes before extraction to prevent problems with static electricity.



DNA extraction from museum specimens- Arthropods -

<Watts et al. 2007



DNA extraction from museum specimens- Plant collections -Material: •Dried leaves, •Other organs, •Seeds, •Etc.

•Surface sterilization of the leave with 1% bleach•Rinced in sterile distilled water•5-10 mg cut into 1mm2 pieces•Grinding in liquid nitrogen + lysis in detergents•DNA protection •from degradation (EDTA to complex Mg, heating, pH)•from oxydation (mercaptoethanol, sodium sulfite)


DNA extraction from museum specimens- Mammals -

•Dried soft tissue

•Skin

•Hair

•Claw

•Hard tissue

Rohland et al. 2004 >



DNA extraction from museum specimens- Skins -

< 253 dried skins < 18 museums in 11 countries.

Stoats (Mustela erminea)

•50% of success with <50 years old � possible amplification with >100 year old samples.•Leather successfully sequenced from a16th century leather sample (nuDNA79bp, mt DNA 177bp

< Martinkova & Searle 2006


DNA extraction from museum specimens- Birds -


•Snips of skin (3 mm2)

•Footpads

•Plucked feathers


DNA extraction from museum specimens- Feathers -


Moulted feathers or pluckedfeathers:

•Easier to sample•No subjected to skin preservationtreatments

A: detail of a posterior viewB: longitudinal cross section

through the feather calamus

1: basal tip of the calamus2: blood clot from the superior

umbilicus

<Horváth et al. 2005


DNA extraction from museum specimens- Fishes -

•Dried scales

<Dermic and epidermic cells outsideand not in the collagen matrix of thescale

Results: up to 50 years, 93% success rate with 200bp

Storing: Best results with paper bagscontaining few scales in a single layer.

•Otolith




DNA extraction from museum specimens- Other -

Dried venom sample�link with voucher specimen

<1995-1998storage at 4°C

starting material: 100–200 mg 500 bp of mtDNA

Success rate: 6/10 samples

ExuviumMouthparts were removedExuvium was crushed using a scalpel blade


DNA extraction from museum specimens- Hard tissue -

+ Act as barrier against autolytic, oxidative and hydrolytic DNA damage (including fluid preserved specimens)


•Décontamination with 20% (v/v) Clorox bleach solution •Decalcification with EDTA•Digestion with N-phenacylthiazolium bromide (PTB) and PK•Washing with water�Ready for classical DNA isolation

•Teeth

•Maxilloturbinates

•Bones

Bone tissue undermicroscope


DNA extraction from museum specimens- Teeth -


Cutting the tooth with a high speed hand held rotary tool before DNA extraction

… or the whole tooth in the lysis buffer

< Rohland et al. 2004


DNA extraction from museum specimens- Ancient DNA and (sub)fossils -


< Austin & Arnold 2006




•Wet samples from water-saturated environment (10000 years, 125bp)

•Frozen samples from permafrosted layers and glaciers (40000 years, 150bp)

(Ex Ötzi, 5000 years old: 10-20 equivalent nucleargenome/g tissue)

•Ancient plant seeds (44000 years)

•DNA from amber inclusions (4-225 millions of years)

< Hebsgaard 2005

•Plant compression fossils

•Ancient bones




Inhibitors:Contaminants are often co-extracted with aDNA from teeth, bones, and mummified tissue (humic acids, fulvic acids, tannins, porphyrin products, phenolic compounds, hematin, and collagen type I)�Repeat silica extraction

In general:•More material (10 g of frozen skin of a mammoth carcass)•Destructive sampling•Final elution ~20µl•Smaller DNA fragments can be amplified <100bp



DNA extraction from museum specimens- Checking aDNA -



DNA extraction from museum specimens- General references (1) -

Jeremy J. Austin, Andrew J. Ross, Andrew B. Smith, Richard A. Fortey and Richard H. Thomas, 1997,Problems of reproducibility - does geologically ancient DNA survive in amber-preservedinsects? Proc. R. Soc. Lond. B, 264, 467-474

J.J. Austin, E.N. Arnold, 2006, Using ancient and recent DNA to explore relationships of extinct andendangered Leiolopisma skinks (Reptilia: Scincidae) in the Mascarene islands MolecularPhylogenetics and Evolution 39, 503–511

Punyasloke Bhadury, Melanie C. Austen, David T. Bilton, P. Jhon D. Lambshead, Alex D. Rogers and Gary R. Smerdon, 2007, Exploitation of archived marine nematodes — a hot lysis DNA extraction protocol for molecular studies, Zoologica Scripta, 36, 1, January 2007, pp93 –98

Matthew D. Dean and J. William O. Ballard, 2001, Factors affecting mitochondrial DNA quality frommuseum preserved Drosophila simulans, Entomologia Experimentalis et Applicata 98: 279–283

M. Thomas P. Gilbert, Wendy Moore, Linea Melchior, Michael Worobey, DNA Extraction from Dry Museum Beetles without Conferring External Morphological Damage, PLoS ONE, 3, e272, Published online




Mehrdad Hajibabaei, Jeremy R. deWaard, Natalia V. Ivanova, Sujeevan Ratnasingham, Robert T. Dooh, Stephanie L. Kirk, Paula M. Mackie and Paul D. N. Hebert, 2005, Critical factors for assembling a high volume of DNA barcodes, Phil Trans. R. Soc. B, doi:10.1098/rstb.2005.1727, Published online

Martin B. Hebsgaard, Matthew J. Phillips and Eske Willerslev, 2005, Geologically ancient DNA: fact or artefact?, Trends in Microbiology, Vol.13 No.5

Márton B. Horváth, Begoña Martínez-Cruz, Juan J. Negro, Lajos Kalmár and José A. Godoy, 2005, An overlooked DNA source for non-invasive genetic analysis in birds, Journal of avianbiology 36: 84/88

Natalia V. Ivanova, Jeremy R. deWaard, Mehrdad Hajibabaei and Paul D.N. Hebert, Protocols for High-Volume DNA Barcode Analysis, Draft Submission to DNA Working Group Consortium for the Barcode of Life

C. William Kilpatrick, 2002, Noncryogenic preservation of mammalian tissues for DNA extraction: an assessment of storage methods, Biochemical Genetics, Vol. 40, Nos. 1/2, 53-62




Mauro Mandrioli, Federica Borsatti and & Lucrezia Mola, 2006, Factors affecting DNA preservation from museum-collected lepidopteran specimens, Entomologia Experimentalis et Applicata, 120: 239–244

N. Martinkova and J. B. Searle, 2006, Amplification success rate of DNA from museum skin collections: a case study of stoats from 18 museums, Molecular Ecology Notes (2006) 6, 1014–1017

Michele K. Nishiguchi, Phaedra Doukakis, Mary Egan, David Kizirian, Aloysius Phillips, Lorenzo Prendini, Howard C. Rosenbaum, Elizabeth Torres, Yael Wyner, Rob DeSalle and Gonzalo Giribet, 2002, DNA Isolation Procedures, Methods and Tools in Biosciences and Medicine, Techniques in molecular systematics and evolution, ed. by Rob DeSalle et al.

Svante Pääbo, Hendrik Poinar, David Serre,Viviane Jaenicke-Despr´es, Juliane Hebler, NadinRohland,Melanie Kuch,1 Johannes Krause, Linda Vigilant, andMichael Hofreiter, 2004, Geneticanalyses from ancient DNA, Annu. Rev. Genet. 38:645–79

Nadin Rohland, Heike Siedel, and Michael Hofreiter, 2004, Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens, BioTechniques 36:814-821




Daniel L. Rowley, Jonathan A. Coddington, Michael W. Gates, Allen L. Norrbom, Ronald A. Ochoa, Natalia J. Vendenderg and Matthew H. Greenstone, 2007, Vouchering DNA-barcoded specimens: test of a nondestructive extraction protocol for terrestrial arthropods, Molecular Ecology Notes, to be published

Evonne P. Y. Tang, 2006, Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collections: Workshop Summary, Editor, National Research Council http://www.nap.edu/catalog/11712.html

Phillip C. Watts, David J. Thompson, Katherine A. Allen, Stephen J. Kemp, 2007, How useful isDNA extracted from the legs of archived insects for microsatellite-based population geneticanalyses?, J Insect Conserv (2007) 11:195–198

About storing techniques, see pdf file « Animal specimens and tissue samples for use in molecular genetic analyses »

About detailed extraction of museum specimens , see pdf file « DNA from archival specimens »

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Molecular genetic investigations in natural history ...jemu.myspecies.info/sites/jemu.myspecies.info/files/nov7_part2... · Molecular genetic investigations in natural history collections---