Thursday, September 18, 2014

DNA Barcoding and killing agents

source INBio Costa Rica
[..] sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA-friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA-friendly protocol for killing. In this study we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). 

This is part of an abstract to a brand-new paper that just appeared in Molecular Ecology Resources. Ever since we started using molecular methods it has been one of the most discussed issues and many researchers started with the question what killing and preservation method should be used to ensure sufficient DNA quality while keeping the potential voucher specimen intact? Chemicals such as ethyl acetate and formaldehyde have been discussed as substances that have the potential to degrade DNA and as such may not be appropriate for the collection of samples for any DNA-based research. Especially formaldehyde represents an issue as killing agent but even more so as preservation liquid. It still is probably the most widely used preservation option we have. It is cheap and provides stable long term storage conditions that keep a specimen intact for future morphological analysis. However, especially over a longer period it will inevitable destroy DNA in an irreversible manner. Colleagues have looked into this issue in a lot of detail simply because most museum fluid collections are using formaldehyde and therefore seem to be lost for any molecular work. 

As for killing methods entomologists are mostly using either ethyl acetate, cyanide or freezing. A molecular biologist would probably always advise to use freezing. No chemical involved would mean no risk for DNA damage and assuming the temperature is low enough it would also help to keep the specimen intact (including coloration). However, in the field very often this is not suitable as you either have to transport the animals alive to the next available freezer or even take a cryogenic shipper with you. Ethanol or other liquids (DMSO-solution, RNAlater) would be an alternative as well and are certainly not DNA-damaging. However, the killing and storage of insect specimens in solutions may not be appropriate for groups where fragile morphological features such as scales on external parts of the body such as the wings may be damaged by wet killing and storage. Furthermore, most insect collections contain pinned specimens and those are in much better shape if they haven't been in any liquid before. 

In this study the colleagues from the University of KwaZulu-Natal in South Africa examined the recoverability of DNA Barcodes from lepidoptera specimens collected using the three common methods described above. Their results are intriguing although currently limited to one insect order. 

All Lepidoptera collected produced DNA barcodes of good quality and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis. 

I am fairly confident that future studies with other arthropod groups will show similar results although I can imagine that issues rather correlate with specimen size or duration of treatment.

1 comment:

  1. Some collectors who use alcohol for killing but preserve their material dry get very good results (morphologically) using acetone. I would be very interested to see how this affects DNA quality.