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Ann Arbor Adventure Part III

Welcome to the final installment of the Joseph Moore Museum’s trip to the University of Michigan. If you didn’t catch the first two posts, scroll below!

Symposium 2017

March 11th saw us once again up bright and early to attend the 13th Annual University of Michigan Early Career Scientists Symposium (say that ten times fast!). This year’s topic was the Ecology and Evolutionary Biology of Phenotypic Plasticity. Phenotypic plasticity is the ability of an organism to change its phenotype in response to different environmental conditions. A phenotype is the physical expression of a genotype, which is an individual organism’s set of genes. Phenotypic plasticity can include behavioral, physiological, and morphological changes that occur over an individual organism’s lifespan in response to new environmental conditions.  

There is a lot of exciting research happening on how phenotypic plasticity evolves and how it affects ecological systems. While we heard a full day of fascinating talks, we’ll just highlight some of our favorites.

Symposium presentation 2017

Dr.  Holly Moeller’s gave a talk entitled Trade, Borrow, or Steal: How acquired metabolism confers phenotypic plasticity. She explored the way different organisms can actually change their metabolism (way of obtaining energy) by taking cellular machinery from other organisms or developing mutualistic metabolic relationships. Marine ciliates, a type of single-celled protozoa, from the genus Mesodinium steal chloroplasts from their prey. They then use these chloroplasts to produce their own energy.  Some even take nuclei to genetically transcribe more phototrophic machinery. Imagine if you could go outside and take some leaves form a tree and use that to gather sunlight for your dinner!

Dr. Ben Parker studied pea aphids, which have an incredible adaption to over-population. Pea aphids usually do not have wings, but if their environment becomes too crowded, they can produce offspring that do have wings. This allows the offspring to fly away to find a roomier home. Some aphids will also produce winged offspring in response to diseased plants, to allow dispersal to better resources. Dr. Parker also looked at the genetic variability in phenotypic plasticity in the aphid population. Some aphids will always produce winged offspring if the conditions are right, while others never do.

 

Aphid

Pea aphids with two offspring (commons.wikipedia.com)

Finally, Dr. Daniel Schwab added some insightful perspectives to developmental plasticity. He explained that as organisms develop, they change in response to internal environmental conditions (such as nutrients and hormones) as well as external environmental factors.  However, organisms are not passive or separate from their environments, but actively change their worlds by building homes or making tunnels. These environments changes that they create can in turn affect their phenotype. For example, dung beetles from the genus Onthophagus have polyphenic horns, meaning the size of their horns change in response to different conditions and relationships with the environment. These include available nutrients, hormones, symbioses with other organisms, and the type of burrows the beetle digs. Thus, the relationship between intrinsic and extrinsic factors is important to fully understand the horn plasticity in the beetles.

After the symposium, we enjoyed a reception back at the Natural History Museum, where we chatted with other scientists and checked out the exhibits. At last, we hit the road back to Earlham. Time for some sleep! And lots of homework…

Poster presentation

Poster for the 13th Annual University of Michigan Early Career Scientists Symposium–Artwork by John Megahan

 

 

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Ann Arbor Adventure! Part II of III

In the afternoon at the Natural History Museum, Adam Rountrey, the Vertebrate Collections Manager, showed us around the collections. In the the paleontology department, we met several U of M students busy carefully packing up ancient bones and fossils for the move over to the new Research Museums Complex. William Sanders taught us about making cradles, which are special containers tailor-made to transport and store a fossilized specimen. Making a cradle is a labor intensive process, especially when you are trying to protect a giant mastodon skull!

William Sanders in paleo

William Sanders standing next to a “cradle”

We then learned about the process of making casts and molds of fossils. First, if the fossil is still embedded in its matrix (rock and sediment), it must be removed and cleaned.  Then, the scientist must decide what type of material to use for the cast. For example, if the scientist wants to study microdamage on the fossil, they might choose silicon, which creates detailed molds but takes a long time to set. Thicker materials dry faster, but do not preserve the same detail. The museum also makes hollow casts, which are much lighter and easier to work with. Hollow casts give more flexibility on how the specimen is displayed. Think about it—as massive whale skeleton, like the one hanging in the Exhibit Museum—would be far to heavy to be safely suspended above your head if it were not hollow inside.

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Casts like these allow guests to touch fossils

We also got a sneak peek into the museum’s incredible imaging lab. Here, they can scan and then 3-D print models of their specimens. While the 3D prints are perfect for displays, they cannot print to the same precision as casts and molds. However, Tom told us that the technology continues to improve.

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The museum’s nifty imaging equipment

After learning how fossils are cleaned, cast, and carried, we descended to the museum’s basement to meet some paleo specimens face-to-face. We saw mastodon skulls, pygmy hippos, and peccaries. Peccaries are hoofed mammals that look like fury pigs, and they lived in Michigan during the Pleistocene. The museum collections has several peccary skeletons that are so well preserved they look like they could be just a few years old. However, these peccaries actually died thousands of years ago, although we do not know exactly when because they have no yet been carbon dated.

Anna looking at pecarry

Anna in Peccary Paradise

We also explored the mollusk collection, which contains over 5 million specimens! Some of mollusk shells are so tiny, hundred fit in a single jar. One of our staff members remarked on the beautiful, chest-high wooden cabinets housing the collection. Thomas Duda, Associate Curator of Zoology, explained that they were specially created for the first collection manager who was a short woman who did not want to climb ladders. We also learned that freshwater clams, euniadids, often look completely different and have different names, depending on if they live in lakes or streams, even though they are closely related.

As our conclusion to our museum visit, we talked with Carla Sinopoli, Director of the Museum Studies Program at the University of Michigan. We told her about the Museum Studies program at the Earlham College, and she explained the certificate program offered by the University. The one-year program is completed at the same time as a student’s main graduate or PhD degree. The program requires four classes and an internship, which can be anywhere in the world!

And at long last, the moment we had all been waiting for…dinner at Zingerman’s! There, we tasted an array of cheeses, made from milk, goat, sheep, and water buffalo milk. After sampling and purchases, we chatted over delicious sandwiches before returning to the hotel and relaxing (in the hot tub!) for the symposium tomorrow. Don’t miss our next blog post on the fascinating conference!

Collection box

Some newly discovered species we saw at the museum…

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Ann Arbor Adventure! Part I of III

Sunny days and snowstorms, seniors madly studying for comps, red-foldered prospectives popping up in classes…it must be March at Earlham College! And with March, comes the Joseph Moore Museum’s annual field trip to Ann Arbor. This year’s trip to the University of Michigan’s Natural History Museum and Early Career Scientist Symposium included six museum enthusiasts—Heather Lerner, Anna Carlson, Lydia Evans, Caroline Wolfe-Merritt, Katherine Sorrows, and Arden Ambrose-Winters.

Upon arrival (well, after a quick coffee stop) Katherine met with the graduate program of the Earth Science Department. Katherine learned about the department’s 5 year PHD program. After two years of courses, students take a qualifying exam to continue with the program. This first part of the exam is written, while part two is an oral summary of the research a student has accomplished to date (even if the research wasn’t successful—whew!).

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Research Museums Complex at the University of Michigan

Meanwhile, the rest of the museum crew explored the University’s brand new Research Museums Complex, located five miles south of the old exhibit museum and collections. We met with EC almun and collection manager Janet Hinshaw, who gave us a tour of the new facilities and the Ornithology (birds) collection. Too bad Janet doesn’t have wings, because the new cabinets are 12 feet tall!

Janet Hinshaw

Janet Hinshaw in the Ornithology Collection

In addition to birds and a few mammals, we also toured the wet collections with Greg Schneider, who is the Herpetology (reptiles and amphibians) Collections Manager. The wet collection contains about 5 million specimens, housed on dozens upon dozens of 15 foot high rolling shelves.

Arden in herp collection

Arden looking at the Herpetology Collections

Greg showed us Earl Werner’s collections from the University’s E.S. George Reserve, a nature reserve which includes 37 natural ponds. The comprehensive, annual collection of specimens from this site has led to a deeper understanding of larval amphibian communities. Another exciting collection we saw are caecilians, which are a type of amphibian that look like snakes. Imagine a snake that feels like a frog!

Specimens with Greg

After saying goodbye to Janet and Greg, we drove back to the Natural History Exhibit Museum to talk with John Klausmeyer, Exhibit Preparator, and Kyra Berman, Associate Director of Education. Anna was prepared with paper, pen and a list of questions about how to successfully redesign JMM’s paleontology exhibit (“a million and one” questions as Anna put it). John was full of helpful tips, from the importance of including touchable objects to how to prevent guests from walking away with said touchable objects!

Museumers with John and Kira

Touching petrified wood! (Left to Right: Arden, Anna, Lydia Katherine, Caroline, John, Kyra)

We also discussed with Kyra how museums can include advocacy in exhibits. Kyra explained this is an important function of museums, but one that must be considered carefully. In order for an exhibit to have lasting power, the exhibit must be relevant now and in the future. One project Kyra is working on is connecting scientists to the public and making science seem more approachable. She organizes opportunities for scientists to hold programs in the museum, as well as organizes informal events like science cafes.

Exhibit hall.pngWhile we would have talked all day with John and Kyra, a packed afternoon still lay ahead of us. Stay tuned for Part II of our Ann Arbor adventure, wherein we explore the paleontology collection, meet some mollusks, and taste fancy cheese!

 

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We have two free events this September!

Building with Biology

 

Learn about the emerging field of synthetic biology and its connections to society. Talk with real scientists and other interested people!

These events are part of a nationwide festival designed to introduce guests to synthetic biology content. They aim to promote informal, two-way conversations between the scientists and visitors about how synthetic biology is interconnected with our society. Free admission to the public!

Should We Bioengineer the Mosquito: A Public Discussion

Thursday September 22, 7-9pm

Center for Science and Technology at Earlham College, Room 300

Register here, space is limited to 60 participants: Registration Form

This is a special opportunity for scientists and the public to interact and learn from each other, and share ideas and opinions about how we want to see these new technologies developed and adopted.

 

Building with Biology Activity Day

Saturday September 24, 1-5pm

Joseph Moore Museum

Enjoy fun, hands-on activities facilitated by Earlham College science students. You can:

  • Design a “Super Organism” to solve a problem
  • Extract DNA from wheat germ
  • Discuss which future technologies you’d support

 

The Building with Biology project is funded by the National Science Foundation and led by the Museum of Science, Boston. Building with Biology kits are developed and distributed nationwide in collaboration with the American Association for the Advancement of Science (AAAS)BioBuilder Educational Foundation, the National Informal STEM Education Network, Science Museum of Minnesota, Sciencenter, and Synthetic Biology Engineering Research Center (Synberc). Events are taking place at over 150 museums and institutions throughout the country from June through September, 2016.

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Debriefing: Notable Quotes

Overheard in the lab:

“Science isn’t all serious.”

“Maybe there was a giant beaver war”–suggestion as to why giant beavers became extinct

“For people who enjoy beavers, this is a sad time.”

“Tiny baby beaver teeth”

“We may be able to use the force, but we can’t keep the lights on”–when the lights in the modern lab kept automatically going off even when we were using the centrifuge

“My fingers are indeed dirty. I will get new fingers.”–said when new gloves were needed

Tare Puns:

“That’s terrible,” “Only a terrorist would do that,” “You’re tearing up our hearts,” “I’m going to tear myself away,” and many more.

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Heather wearing a beaver

 

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qPCR, Strawberries, and Kids

Heather came in on Saturday to check on our DNA library that we had left on the heat block overnight. She discovered that the liquid at the bottom of the tubes was brown. It seemed like some of the liquid had evaporated. This was not good, so she vortexed them and hoped for the best.

We spent Tuesday morning washing our beads. The beads are magnetic and can attach to the DNA. Using magnetic beads is great for ancient DNA because they prevent tiny DNA fragments from escaping. Washing the beads was very visually pleasing because they were brown as opposed to clear. Moreover, we got to use the magnetic rack, which has magnets that pull the beads to one side of the tube. We would start by adding a wash buffer to the tube with the magnets and the solution would be brown. We would then place the tube on the magnetic rack and watch the solution gradually become transparent (video) with a small ball of beads huddling by the magnet.

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Emily showing of the magnetic rack. The black circles are the magnets.

In the afternoon we made a qPCR to see if our DNA library was successful. Unfortunately it was not. The samples started amplifying at around 25-28 cycles and looked no different than the qPCR blank. Last year the samples started amplifying at 20 cycles. It is possible something went wrong with the qPCR machine, but we guessed that the library didn’t work because of Saturday’s discovery of the brown liquid and evaporation. Luckily, a heated lid for the heat block would prevent evaporation and the formation of brown liquid. We are ordering one now.

On Wednesday afternoon we did a DNA demonstration with Miller Farm’s kids’ camp. We showed 7 kids ages 5-8 strawberry DNA and let them help us mash the strawberries. We added dish soap and Gatorade to break down the strawberry cells and then pineapple juice, which has enzymes that break down proteins. Finally we added cold alcohol to separate out the DNA. After letting the test tube sit, we could see a layer of white, goopy stuff that was the DNA.

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The white layer is strawberry DNA

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Using transfer pipettes for pineapple juice

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Extracting DNA from cheek cells

We then let the kids see their own DNA. After swishing Gatorade in their mouths for two minutes, they spit into test tubes. We added dish soap and cold alcohol and showed the kids how to use a transfer pipettes to add the pineapple juice. The kids loved using the transfer pipettes. While waiting for the DNA to separate out, we had the kids make DNA bracelets. They got to choose a DNA sequence from options such as human, butterfly, cobra, flesh-eating microbe, and cockroach DNA. They had to read the sequence and put colored beads corresponding to A,T,C, and G on one string and then figure out the corresponding base pairs to put on the other string. After they finished the bracelets their DNA had separated from the Gatorade and soap. We put the DNA in a little test tube and attached the test tube to a string to make a necklace. It was a nice change of pace to be outside with the kids, and the kids seemed to enjoy learning about DNA.

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DNA bracelets

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Emily helping with bracelets

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Shear Joy

On Tuesday the 24th, we tried to shear our modern beaver DNA into pieces about 150-200 base pairs long so that we could continue making our baits. Shearing DNA involves putting the sample into a machine called a sonicator that uses ultrasonic waves to break up the DNA. Think of an ultrasonic jewelry cleaner, but much more powerful. The sonication machine was brand new, so we didn’t know for sure how long to run it for or what intensity to use. Our first try was a conservative 3 minutes at 30% intensity, which worked nicely; the DNA got down to about 900 base pairs. After this, we ran into trouble. We sheared the DNA again to get it down to the size we wanted it, but it seemed like no matter how long or on what intensity we ran the sonicator, we couldn’t get the DNA below about 300 base pairs. We ran the sonicator five times on Tuesday, and after calling the company for troubleshooting on Wednesday morning, we ran it three more times. Finally we got the DNA down to about 200 base pairs. On Thursday, we used the sheared DNA to finish creating the baits. We used the Nanodrop machine to see how many baits were present, and the result was incredibly high. So high, in fact, that we were very suspicious about how true that was. We plan to run a more accurate test later to see how successful we actually were.

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The sonicator  makes an ear-splitting noise

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Up close and personal with the sonicator. We had to maintain the water level exactly at the level of the samples in the tubes. If it got to high, the samples would jump up the sides of the tube and escape shearing. 

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Shearing is a three-person job

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Cats and Korlević

Doing research involves a lot more than just lab work. We also read a lot of papers from people who have done foundational research that we’re building from. Two such papers are “Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA” by Gansauge and Meyer (2013), and “Reducing microbial and human contamination in DNA extractions from ancient bones and teeth” by Korlević et al. (2015).

Gansauge and Meyer introduce and test a new method for improving the recovery of ancient DNA for DNA sequencing. In this method we take ancient DNA and add little bits of known DNA sequence to them that we can use later to copy the ancient DNA and sequence it. Many copies of the ancient DNA (with attached known sequences) are called a DNA library. In ancient DNA, many strands of DNA are often damaged and broken. DNA is present as two attached copies. Previous methods required both strands to be present and intact, but if they’re broken they get lost during processing. The thing that differs in this method versus others before it is that this method uses single-stranded DNA instead of double-stranded, so much more of the DNA is sequenceable. Another cool thing about this method is that they bind the DNA to magnetic beads so that they can be sure they aren’t losing any of it.

Korlević et al. talk about the contamination present in ancient DNA and how to decrease it. We don’t often think about it, but literally everything is covered with bacteria, and bacteria all have their own DNA. This means that any sample will be contaminated with bacterial DNA, and often with human DNA from handling. Korlevic et al. test three different methods of getting rid of the extra DNA: (1) using bleach on the samples, (2) incubating the samples in a phosphate buffer, and (3) re-extracting the DNA from the sample multiple times. Bleach was the best at getting rid of the contaminating DNA, but it also got rid of a lot of the target DNA, so it isn’t a good method for rare or precious samples. The phosphate treatment turned out to be the safest, since it preserved all of the target DNA, but it also didn’t get rid of as much contaminating DNA. The re-extraction method didn’t help significantly, and even made the contamination worse in some cases.

Reading papers like these is important for understanding the work that came before ours, and putting our own work into context. The papers can be difficult to read and understand, but reading them is an essential part of the research process. It’s also not too bad when you get to pet kitties while reading!

 

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Maren and me at Heather’s house reading papers with her cat, Legolas.

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Playing with fun machines

 

On Wednesday Chris Smith kindly showed us how to use fluorescence to determine how much mitochondrial DNA we amplified last week. This is done on a Quantitative PCR (qPCR) machine using picogreen.

When we ran our reaction, both long products and unintended smaller pieces amplified. We needed to separate the unwanted smaller products out from the longer ones. To run all nine samples using four primer pairs, we had to run two gels. Gels use charge to push DNA through the gel. DNA is negatively charged, so applying a negative charge forces the DNA to move through the gel. Bigger pieces of DNA will get stopped in the gel earlier and smaller pieces will travel further, so when looking at the gel after it is finished running, we can determine which bands are bigger pieces of DNA.

I enjoy making and running gels because we can finally see what we are dealing with. We mixed 1 g of Agarose in TAE, and then heated the mixture up in a microwave. Apparently Agarose is quite viscous and will burn you and refuse to come off if it touches your skin, so Heather kindly didn’t let us handle it while it’s heating. After the Agarose dissolved we poured the gels and learned the hard way to not assume that they’re set before trying to yank them up.

Unlike yesterday, we ran the gels at the recommended 60 mV so that our bigger chunks of DNA wouldn’t be pushed through the gel too quickly or get heated up and damaged. After an hour we read the gels under UV light and they both showed DNA, which was a good sign.

However, we still needed to get the DNA out of the gels. The Gel-Doc has a UV light underneath the tray that the gel sits on, so the DNA bands are illuminated. We cut the brightest bands (the DNA) out with a razor and put them in a tube. This was dicey because we had to wear sunglasses and a scratched or damaged UV shield so that the UV light didn’t burn us up. This made spatial reasoning more difficult. The bands were completely surrounded by the gel, so we had to cut the gel away from all six sides of the band while moving quickly enough so that the DNA wouldn’t be damaged by the UV light. While one person cut out a band, another would tare a labeled tube and then hold it out for the cutting person to put the band in, then weigh and record the tube, and finally return for another band and repeat the process. We had an exchange of tare puns with Peter’s group. There were some terrible ones and we finally had to tear ourselves away.

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Cutting out the bigger bands from the gel using the guidance of the Gel-Doc’s inner UV light. 

After we got the bands in the tubes with nobody burned from the UV light, we added a Guanidium buffer (more Guanidium! Yay!) to each tube and centrifuged the tubes. We ended the day by eluting the DNA cut from the gels and loading the qPCR machine. Unfortunately, we failed to retrieve the data but the next morning Chris saved the day and exported our data. Luckily the data was there; we had just messed up getting it off the computer. Unfortunately, the data wasn’t great: some of the samples that should have had similar values were drastically different and our standard curve was not a nice line. We figured that our pipettes were off and got permission to use Chris’s pipettes for the next time. We re-ran the picogreen assay and got improved results: our standard curve had an r^2 of 0.89 and the values were more similar across primers. Primers A-D had reasonable amounts of DNA but Primers E-H had extremely low amounts.

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Plastic knuckles for ensuring that the caps on the tubes are secure for the picogreen assay! 

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In which we calculate Molarity and get our First Results

The past few days have gone by quickly! Apparently, we did six days of work in our first five-day week, which was satisfying to learn. By the end of last week we had made our binding buffers, added them to our samples, and centrifuged them, and set up our Long Range PCRs.

However, Friday was the 13th, so some mishaps were to be expected. The Binding Buffer called for 39.8 g of Guanidine, a solid that looks like sugar that’s been sitting in someone’s china cabinet for 10 years. Guanidine becomes Guanidium in water, although it is notoriously hard to dissolve. Even still, 39.8 g seemed like too much, so we ran around and did math until we figured out that it was supposed to be 23.8825 g. Today, Heather found out that the protocol we had been using had miscalculations, and that we were correct to use 32.8825 g of Guanidine. That was a relief!

We have a vortex in the Ancient lab, but it strangely didn’t come with a plug. The cord just ended in a series of wires. Heather bought a plug and we wired it ourselves to no avail. In the end, we used our inner human vortexes and it was fine. At the same time, our pH meter didn’t want to calibrate, so Emily spent a long time fiddling with it. We needed to get the pH of the sodium acetate to 5.2. Eventually, we got close enough.

On Monday, we had a shorter day. In the morning, we made several Wash Buffers in the Ancient lab. Everything went smoothly. The only hiccup was when we discovered our SDS was 10% instead of 20%, so we compensated by using twice as much and adjusting the amount of water added to the solution. After lunch, we went up to the modern lab and made a gel to run the Long Range PCRs on. Previously, I had only used pre-cast gels, so it was satisfying to make the gel ourselves. We pipetted our DNA into the wells as quickly as possible so that the DNA couldn’t leak out. After the gel was finished running, we looked it on an imager. The gel (below) showed excellent amplification of partial mitochondrial genomes for the modern beaver!

LR PCR Gel 16 May 2016

Gel of Beaver Mitochondrial DNA: There are two rows. The two long smears on each end of the top row and in the second to last lane of the bottom row show a DNA ladder with known (short!) sizes of DNA fragment. The other bands on the top row are ~11,500 bps of DNA, while the bottom row has ~6,000bp fragments of DNA. 

Now we can go forward turning those sequences into baits to fish out ancient DNA.

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