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The art of getting DNA out of decades-old pickled snakes

The art of extracting DNA from decades-old inlaid snakes

Part of the Field Museum’s underground “bunker” of reptile specimens. Credit: Field Museum

Chicago’s Field Museum, two stories underground, has a secret bunker. The sub-basement Collections Resource Center is home to millions of biological specimens that scientists around the world can use in their research, including countless bottles and jars of pickled fish, lizards, and snakes arranged like a library. Many of these specimens are decades or even centuries old, almost perfectly preserved by a combination of formalin and alcohol. But the process that preserves tissues often destroys or makes obtaining DNA very difficult for modern studies, which is bad news for scientists studying genetic relationships between organisms. A new study in Frontiers in Ecology and Evolutionhowever, is uncovering new approaches to obtaining and maximizing useful DNA from decades-old brined specimens, using these techniques to solve a long-standing mystery about a small snake from the island of Borneo.

“As a true crime buff, it reminds me of how people didn’t take DNA samples when a crime was committed in the 1960s, because no one could have predicted that one day you would be able to use DNA samples to find out who committed a crime” , says Sarah. Ruane, Field Museum assistant curator of herpetology and senior author of the study. “These older museum specimens are sometimes the only available examples of a species, but they weren’t preserved with DNA in mind — this article is about how we can squeeze out all the possible information.”

The project grew out of Justin Bernstein’s dissertation research when he was Ruane’s student at Rutgers University-Newark. “My primary study involves a group of snakes, called homalopsids or mud snakes, that live in South and Southeast Asia, New Guinea and Australia,” said Bernstein, the lead author of the paper. “They’re really fascinating; they live in muddy, aquatic environments, and there are 56 species of them. We use DNA to study their evolutionary history, to try to describe new species and to learn what happened to these groups.” over tens of millions of years that led to the diversity we see today.”

The art of extracting DNA from decades-old inlaid snakes

Alcohol-preserved specimens of Hydrablabes appear in the Field Museum’s collections. Credit: Josh Mata, Field Museum

For this article, there was one animal in particular that Bernstein and Ruane were trying to place in a family group, called a six-foot greenish-brown snake. Hydrablabes period, also known as the small-eyed olive snake. It is found on Borneo, a large island east of mainland Malaysia and northwest of Australia that contains parts of Malaysia and Indonesia and the entire nation of Brunei. Based on its appearance, scientists had suggested two different families that it may be part of. Analyzing the DNA could reveal his closest relatives and thus his family, but that was easier said than done. “About half the diversity of mud snakes and stuff” Hydrabla berries There are known vouchers from older specimens that have been chemically treated, and this chemical treatment breaks down the DNA,” says Bernstein.

Part of the process of preserving an animal in alcohol is “fixing” it with a substance called formalin, a solution of formaldehyde gas and water, which makes its tissues rubbery and stiff. Unfortunately, the DNA in his cells is also changed. “It does something called crosslinking, which binds the DNA,” Ruane says. “If you want to study its DNA, you have to undo the DNA or try to squeeze it out of those crosslinks.”

Studying the olive-colored small-eyed snake involved taking small samples of liver tissue from some of the few specimens in the United States, both from the Field Museum’s collections. One from 1964 and the other from a collection trip in 1993 by Robert Inger, then curator of The Field, and his wife Tan Fui Lian.

The art of extracting DNA from decades-old inlaid snakes

Lead author Justin Bernstein visits the Field Museum’s reptile collections. Credit: Sara Ruane, Field Museum

Such old specimens required new laboratory techniques. Normally, obtaining DNA from a tissue sample involves adding digestive enzymes that break down the tissue, leave the DNA behind and heat it to 130°C for several hours. “We had to change the way we got the DNA out by making it hotter longer and using more of these digestive enzymes,” Ruane says. These more extreme preparation methods were effective for other snakes in previous studies, but the resulting genetic analysis still contained many gaps for Borneans Hydrabla berries snake specimen.

The art of extracting DNA from decades-old inlaid snakes

Lead author Justin Bernstein works with snake specimens at the University of Kansas Biodiversity Institute & Natural History Museum, using morphological data to investigate the diversity of homalopsid snakes. Credit: Justin Bernstein

“The chemicals used to preserve the snakes cut their DNA into shorter pieces of code, making them difficult to compare to longer, more complete genes from other specimens,” Bernstein says. “The first software I used made it difficult to understand how much fragmented DNA there was in the study specimens, but switching to other software that visualized the pieces of genetic code made it easier to see where there were problems.” And even the smaller, more fragmented bits of code can be added to larger, published datasets to help build an evolutionary tree.

An important aspect of this article for Bernstein was to be transparent about the difficulties of using older specimens and the troubleshooting required to study them. “I wanted to show scientists that you can still work with these specimens, it just takes some detective work,” he says. “On a broader level, the study really shows how to use the data you acquire and how to combine it with previously published data sets to explore some really cool hypotheses.”

The art of extracting DNA from decades-old inlaid snakes

One of several decades-old snake specimens from the Field Museum used in this study. Credit: Sara Ruane, Field Museum

As for the Bornean snake at the heart of the study, the researchers were able to determine that it is a member of the family Natricidae, which includes distant relatives such as the North American garter snakes. That may not seem like a big deal, but “knowing that a particular species is part of a particular group can tell us a lot about biogeography and about how life on Earth has changed over time,” Ruane says.

And aside from the study of snakes, she notes that overall, “this project underscores the importance of museum collections because you never know what you might learn from specimens in the future.”


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More information:
Maximizing molecular data from low-quality liquid-preserved samples in natural history collections, Frontiers in Ecology and Evolution (2022). DOI: 10.3389/fevo.2022.893088

Quote: The art of extracting DNA from decades-old pickled snakes (2022, June 30) retrieved June 30, 2022 from https://phys.org/news/2022-06-art-dna-decades-old-pickled-snakes . html

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