Policies and best practices Policies and best practices for molecular analysis of for molecular analysis of

museum specimensmuseum specimens

Peter T OboyskiPeter T OboyskiEssig Museum of EntomologyEssig Museum of Entomology

UC BerkeleyUC Berkeley

“All of this has happened before,

and it will all happen again.”

ECN meeting 1995 – Jim Whitfield

What molecular analyses?

• DNA sequencing of the specimen• Parasites, pathogens, or pollen• Stable isotope analyses

The Problem

• With the advances in molecular techniques and molecular systematics we can expect to see more loan requests for molecular analyses– How to respond?

• Destructive sampling is in conflict with preservation– At the discretion of the curator– Recent methods do not require grinding tissues

• Policies need to be in place to respond to requests

Policy Recommendations I:Necessity

• Is there just cause to use museum specimens?– Historical analyses

– “Impossible” to access collecting locations

– Local extirpation or extinct species

Xerces blue

Necessity: Availability of specimens

• Long series are no problem– What about types or rare specimens?

• British Museum microlepidoptera types– New policy to extract DNA during genitalia preps

Policy Recommendations II:Methods

• Does the researcher have the proper facilities and methods to likely recover DNA?– “Clean room” or “Ancient DNA” room

– Mitochondrial vs. Nuclear DNA

– Fragment length of gene region to sequence• Single Nucleotide Polymorphisms (SNPs)

– Sanger sequencing vs. Next Generation

– Extraction method• Phenol-chloroform extractions vs. Qiagen® kits

Policy Recommendations III:Qualifications

• Does the researcher have the proper background and experience for success?– Experiment with other specimens (not mine!)

– Letter of reference from advisor or DNA facility

– Read between the lines of the proposal/methods

Other considerations

• Scientific merit of the project

• Specimen history: kill method, drying, storage

• Specimen identification: “user beware”

• Funding & time line

What gets returned?

• All remaining tissues & all labels

• Return an aliquot of extract with specimen?– Extracts must be stored in a (long-term) “tissue bank”

– What about costs of storing tissues/extracts?

© Natural History Museum

What about the DNA?

• GenBank accession required (within one year?)

• “All rights reserved” to originating museum

• Should the researcher also return sequence data?

• Do museums have databases to manage these data?– Link GenBank accession numbers with catalog numbers

– Metadata: extract methods, concentration, location, etc.

– DNA sequences for each gene

GenBank Nucleotide Search (BLAST)

Non-invasive DNA Extraction Techniques for Parasitic Hymenoptera

Jeremy C Andersen and Nicholas J MillsUniversity of California Berkeley

Questions

• Can non-invasive techniques be used for parasitic Hymenoptera?

• Concerns:– small body size– delicate

• What are effects of size and age of specimen?• Can DNA extracts be used for PCR and sequencing?

Methods• Selection criteria for Braconidae from Essig Museum of Entomology

1) Species differing in adult size2) Wide range of ages3) Large number of specimens

• Atanycolus longifemoralis 15 specimens 1931 – 1981

• Meteorus trachynotus 9 specimens 1914 – 2009 *

• Trioxys pallidus 12 specimens 1959 - 1993

DNA Extraction

• Followed methods and protocols outlined in Gilbert et al. 2007– Slight modifications to several steps– E-mail: jandersen@berkeley.edu for protocols

and/or materials list

• DNA yield was measured with a NanoDrop spectrophotometer

• Sequenced 28S and COI genes (long & short)• Specimen dried, weighed, and remounted

Results

A. longifemoralis~ 4 mg

M. trachynotus~ 0.2 mg

T. pallidus~ 0.01 mg

Log(

DN

A c

once

ntra

ion)

0

1

2

3

4

5

6

7

p = 0.019

Age

Effect of Specimen age on outcome of PCR (28S)1

0

Discussion• Successful extraction of DNA and PCR

amplification from specimens back to 1940• Two markers differed in their success rates

– Species and Age

Comments• Discoloration of Body

– Reduced percentages of chemical SDS worked for new specimens (Meteorus 2009) with less discoloration

• Weighing and general handling of insects caused most damage

• Primer design is extremely important

• Nanodrop readings (phenol and DNA similar)

Useful LiteraturePääbo, S. (1989). "Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification." Proceedings

of the National Academy of Sciences 86(6): 1939-1943.

Whitfield, J. B. and S. A. Cameron (1994). "Museum policies concerning specimen loans for molecular systematic research." Molecular Phylogenetics and Evolution 3(3): 268-270.

Hafner, M. S. (1994). "Reply: Molecular extracts from museum specimens can—and should—be saved." Molecular Phylogenetics and Evolution 3(3): 270-271.

Whitfield, J. B. and S. A. Cameron (1994). "Authors' response to Hafner." Molecular Phylogenetics and Evolution 3(3): 271-272.

Thomas, R. H. (1994). "Molecules, museums and vouchers." Trends in Ecology & Evolution 9(11): 413-414.

Whitfield, J. B. (1995). "Museum loan and voucher specimen policies for molecular systematics research." Insect Collection News 10: 3.

Phillips, A. J. and C. Simon (1995). "Simple, efficient, and nondestructive DNA extraction protocol for arthropods." Annals of the Entomological Society of America 88(3): 281-283.

Golenberg, E. M., A. Bickel, et al. (1996). "Effect of highly fragmented DNA on PCR." Nucleic Acids Research 24(24): 5026-5033.

Whitfield, J. B. (1999). Destructive sampling and information management in molecular systematic research: an entomological perspective. Managing the modern herbarium: An interdisciplinary approach. S. Byers and D. Metsger. Ontario, Society for Preservation of Natural History Collections and Royal Ontario Museum: 301-314.

Rohland, N., H. Siedel, et al. (2004). "Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens." Biotechniques 36(5): 814-821.

Gilbert, M. T. P., et al. (2007). "DNA extraction from dry museum beetles without conferring external morphological damage." PLoS ONE 2(3): e272.

AcknowlegmentsAcknowlegments

Jeremy Anderson (UC Berkeley)

Jim Whitfield (Univ. Illinois)

David Furth (NMNH/SI)

James Cokendolpher (Texas Tech)

Katrina Menard (Sam Noble Oklahoma MNH)

Chris Marshall (Oregon State University)

James Liebherr (Cornell University)

Doug Yanega (UC Riverside)

Kipling Will (UC Berkeley)

Kevin Tuck (Natural History Museum)

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