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The Tephritid Barcoding Initiative (TBI) is trying to barcode museum vouchers before going to the added expense of collecting and identifying new specimens.  A preliminary study has shown that success rates for PCR of full-length barcodes drop off for specimens older than 10 years.  Using internal primers doubles the success rate for specimens between 10-20 years old but it's still only 50% successful.

I asked people at the Biodiversity Institute of Ontario (BIO) in Guelph and the Laboratories for Analytical Biology (LAB) at the Smithsonian if they have any advice for improving the DNA recovery of older insect specimens.  Here are there replies:

Natalia Ivanova at BIO pointed us to the attached publication of a protocol developed at Guelph.

Bob Hanner at BIO said:  

I dont have any easy answers but one area which appears promising for the concentration of forensic samples and/or dirty environmental samples is the
Boreal Genomics' Synchronous Coefficient of Drag Alteration, or SCODA, system.
This is something that is likely to payoff - but the instrument itself isnt
cheap. Colleagues at the University of Waterloo are reporting spectacular
results using a prototype instrument that runs only one sample at a time, but
from what I understand the company is working to develop a machine that can
handle multiple samples...

Lee Weigt at LAB said:  

I think we have seen very similar results with mosquitos - 10-15 years are far more successful than 20+ years.  First thing to try is better mining of the collections to preferentially get recent material instead of older material and minimize the

next is mini-barcode regions if primers can be developed, but that is more costly in both labor and consumables.  Most expensive, least tried is to allow the sheared DNA to be used, but to multiplex on a next gen sequencer - this is one of the true
values of next gen to DNA barcoding - cost a little more, but probable yield is
complete mt genomes, not just COI.  Worth a stab and a try if someone can get the $15-$25K to
play with...for a plateful of samples.

Amy Driskell at LAB said:

Lee's right, of course. But we've seen that tweaking PCR conditions (really hammering away at it and trying just about everything) - types of Taq, additives, concentrations of everything, cycling profiles - can
increase amplification success. Andrea and I have been hitting the old
mosquitoes pretty hard the last few weeks and have seen some improvement. I'd
be happy to share what we've found out. Yvonne Linton apparently has even
better success with old bugs and I hear she has a new PCR technique. She also
generally amplifies the CO1 in 2 or more smaller pieces.


I think, in general, the problem is not small amounts of DNA, but high amounts of PCR inhibitors. This is what the forensics people seem to believe. But, there's no silver bullet to fix the problem that we've been
able to find.


That's all I know right now. It's very frustrating and success will be expensive in these older specimens.

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Something else to consider is a nested PCR approach. I found it very helpful when tweaking PCR conditions just won't yield a product detectable by agarose gel electrophoresis with ethidium bromide staining. Yet an aliquot from such reaction can make a great template for amplification of shorter fragments. Just be careful, chances of recovering a contaminant sequence also increase.
I've been using the nested PCR and mini-barcode approaches in combination for a few years now on museum specimens from 20-50 years old. Yes one gets quite a few contaminants but we throw out all singletons and only "believe" sequences that are confirmed by sequences from conspecific specimens. My lab's success rate with these older specimens has been ~70%. The key of course is developing good primers. I have developed degenerate PCR primers which amplify two fragments of ~330 bp each, and one internal primer for each fragment which is used for reamplification. Final products are ~310 bp each and overlap each other by ~60 bp. The primers work well in Lepidoptera, Coleoptera, Hemiptera and I wouldn't be surprised if they worked well in flies too though we haven't tried many other groups. Manuscripts being written up at the moment. I can send details to anyone interested...


I am working on Insect barcoding with wide range of order. I am  facing problems in amplification of coleoptera and Hemiptera. Could you please help me or suggest me the primer for the order which you have mentioned above?

I have read your posts about the DNA from (tephritid) pinned specimens with interest.
We here at the africamuseum (RMCA) in Belgium have quite some experience with DNA amplification from old & pinned specimens. We have published a few primers for tephritid fruitflies (all tailed for ease of sequencing downstream) entitled: "Recovering full DNA barcodes from natural history collections of Tephritid fruitflies (Tephritidae, Diptera) using mini barcodes" This may be helpfull, however we did not test if they could be used in a nested PCR.
I agree that amount of DNA from specimens is not a problem, however DNA from pinned specimens (especially abdomens) is very degraded. it takes these specimens a rather long time to dry out properly.
Very specific primers may also help. Platinum taq as better enzyme works but not spectacularly with these types of specimens. If you have any other questions or need help do not hesitate to contact me.



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