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University of Guelph Researchers PCR-Amplify Specimen DNA from Preservative Ethanol


By Ben Butkus

Many a drinker of mezcal, the tequila-like alcoholic beverage made from agave plants, has been warned not to eat the worm at the bottom of the bottle.

According to researchers from the University of Guelph, however, if you drink mezcal at all you will still be ingesting the worm, or at least its DNA — enough of it, in fact, that it can be PCR-amplified and sequenced.

The findings may be important for practical applications such as high-throughput, non-invasive genetic analyses of samples that are scant or require preservation; and improved sample preparation for next-generation sequencing, Mehrdad Hajibabaei, lead researcher on the study, told PCR Insider this week.

In a paper published in the March issue of BioTechniques, Hajibabaei and colleagues at the University of Guelph's Biodiversity Institute of Ontario described their procedure for obtaining amplifiable quantities of DNA from the "worm" (actually the larvae of the agave butterfly, Hypopta agavis) in a bottle of Monte Alban brand mezcal.

Hajibabaei said that his lab at the Biodiversity Institute of Ontario does a lot of next-generation sequencing. "The field of sample prep is serious in that every sequencing study or genetic test goes through some sort of sample prep," he said.

In particular, researchers there work with intact animal or plant specimens — sometimes collected from the field in remote locations. "When the samples came in ethanol jars … we started to detect DNA from different organisms that we did not expect to see," Hajibabaei said.

"We started wondering why we were seeing these patterns of DNA from organisms that we didn't put in the mixture itself," he added. "So we went back to the ethanol jars that contained the samples, and started analyzing the ethanol. I thought if the ethanol is untouched and just carries these samples; and we haven't done any procedure to get the DNA out of the cells; then there must be something happening to provide this DNA."

To explore their hypothesis, the researchers chose a "conventional and kind of cute" example that people could relate to: the larvae in mezcal. But there was another good reason for using the liquor — at about 40 percent alcohol content and with many impurities from the distilling process, it is far from a perfect preservation medium. Thus, the researchers figured that if they could amplify DNA from mezcal, they could certainly do so from lab-grade ethanol.

As described in the BioTechniques paper, they evaporated 50 mL of mezcal, dissolved the residue in water, passed the solution through a Qiagen spin column to remove PCR inhibitors, and re-dissolved the DNA in water. They applied a similar process to freshly collected and archival specimens stored in 95 percent ethanol, but left out the spin column step, which was not necessary for these samples.

Using standard DNA barcoding protocols and primers, they then amplified 130- and 658-base fragments of cytochrome C oxidase 1 from all the samples; and amplified nuclear ribosomal 28S rDNA from freshly collected insect specimens. They performed PCR using pre-mixed PCR plates and Platinum Taq polymerase from Life Technologies' Invitrogen business; and sequenced on a Life Tech Applied Biosystems 3730xl sequencer.

Sure enough, they were able to PCR-amplify and sequence DNA from all samples, including the mezcal. They compared their results to genomic DNA extracted from the larval tissue using a commercial kit; as well as to an existing library of butterfly DNA barcodes.

Although they were not able to amplify a full-length DNA barcode of about 650 bases from the mezcal, they did amplify this fragment from a tissue sample obtained from the worm. The researchers wrote that they believe the DNA obtained from the mezcal had been released from the larvae's body and had degraded over time.

"It's not extracting a lot of DNA, but then you don't need a lot of DNA to do PCR these days," Hajibabaei said. "It's good enough that you can do 50 PCRs. We are now investigating this to try and find out the dynamics and timing of this DNA release. But we have so far worked on old samples, plants, different animals, fish tissue — everything we've thrown at this system worked fine with PCR and sequencing."

One caveat to the approach, Hajibabaei added, is that it is likely beneficial to disturb the sample as little as possible in order for it to work. "Touching and disturbing [the specimen] is something you don't want to do in this situation, because if you do, you start releasing proteins and lipids into the mixture, and later on you will need some sort of purification step," he said.

This is in sharp contrast to traditional DNA extraction and purification kits, in which a sample is typically completely homogenized prior to any chemistry being performed. "We're trying to simplify that," Hajibabaei said. "Just grab the DNA in the preservative medium. I think this can have a lot of application in next-gen sequencing work and the sample prep that is involved with those machines. Also, this is very easily amenable to automation."

It is possible, but not likely, that the particular polymerases used by the researchers contributed to the technique's success, Hajibabaei added. "Obviously the Platinum [Taq], and these spiced up Taq polymerases are good in certain situations, and can help," he said. Just to be sure, the lab is currently testing the method with other PCR protocols, and may have additional data in a few weeks, he added.

Now, the lab is discussing its results with colleagues to find out what applications the method might have. So far, suggestions include next-gen sequencing sample prep; forensics; biological field work; and diagnostics using excise tissue samples — basically any situation where researchers would prefer to keep a sample intact but still obtain DNA for testing.

"I have contacts saying, 'OK, we can't take ethanol into the field, but maybe we can take a bottle of vodka,'" Hajibabaei said. "In remote areas, these types of logistical issues are important."

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