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DNA Recovery from older pinned insects
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...
http://www.borealgenomics.com/tech.html
problem.
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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Permalink Reply by Evgeny V. Zakharov on -
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.
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Permalink Reply by Andrew Mitchell on
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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...
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Permalink Reply by Floris Breman on -
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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