Policies and best practices
for molecular analysis of
museum specimens
Peter T Oboyski
Essig Museum of Entomology
UC 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 Mills
University 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 size
2) Wide range of ages
3) 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
3
2
7
1
0
6
Log(DNA concentraion)
4
5
Results
A. longifemoralis
~ 4 mg
M. trachynotus
~ 0.2 mg
T. pallidus
~ 0.01 mg
Effect of Specimen age on outcome of PCR (28S)
1
p = 0.019
0
Age
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 Literature
Pääbo, 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): 271272.
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.
Acknowlegments
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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