Pooling already extracted dna?

Pooling already extracted dna?

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I had ethanol precipitated a large amount of DNA (2ml) and had to split the sample in half to spin down because only the microcentrifuge has the correct rotor to spin that fast. I want to get as concentrated of a DNA sample as possible. Is it possible (correct) to combine the resuspended DNA? I wanted a total of 30 ul of extracted DNA. Could I resuspend each of the two sample in 15 ul and then combine them?

What is the best way to go about this. I realize just keeping them separate might be the best option. I also realized I could've lost DNA when transferring the sample to a smaller tube. I did this because I don't believe it is correct to spin for longer at a lower speed…

If it were me…

  • You can resuspend the two separate pellets (A and B) in 10ul each.
  • Collate in one tube (add A to B).
  • Use the remaining 10ul from your desired volume to 'wash out' tube B and add the wash to tube A.

So long as the DNA is from the same sample this is a perfectly reasonable step that should not result in a substantial loss.

New Origin Story for Gross Blobs That Wash Up on Beaches

DNA evidence shows that jetsam ambergris comes from sperm whales.

Every so often, a fatberg-esque blob of material called ambergris washes up on a beach.

These lumps, used to make perfume, can be worth thousands of dollars in countries where it is legal to collect them. Historically, hunters have trained dogs and even camels to sniff out ambergris.

Where it comes from has been less clear. Modern scientists knew that ambergris could be found within the bodies of sperm whales, but they weren’t sure about those pieces of jetsam found by beachcombers. But in a study published Wednesday in Biology Letters, researchers have extracted sperm whale DNA from washed-up ambergris, which is especially impressive because the studied samples floated adrift for years, aging under salt, sea and sun. The research could contribute a new understanding about the enigmatic substance and the endangered creatures that make it.

“The discovery that ambergris yields such good DNA preservation opens up new opportunities for studying both the use of this precious raw material and whale biology,” said James Barret, an archaeologist at the University of Cambridge who did not participate in the research.

Fresh ambergris smells fecal, musty. But once it ages, its scent is compared with fine tobacco, or the wood in an old church. The mother of the Abbasid Caliph Al-Muqtadir would melt it in oil Catherine de Medici wore it in scented gloves and ambergris has anointed English monarchs since 1626, including Queen Elizabeth II.

But where did it come from? That was one of the medieval world’s grossest natural mysteries. Marco Polo’s travelogue said it had something to do with whales. That hunch was confirmed by whalers in New England in the 1700s poking around with spades in the rectums of dead sperm whales.

But since whale populations were decimated, much remains unknown about ambergris, and especially the origin of jetsam ambergris. Its chemical composition differs from ambergris that can no longer be dug out of protected sperm whales.

Ruairidh Macleod, an undergraduate biology student also at Cambridge, led the research. Steven Rowland, a chemist who has collected and analyzed jetsam ambergris, including chunks that may have been at sea for over a thousand years, provided the samples, which were picked up on beaches in Sri Lanka and New Zealand.

The team also turned for assistance to the lab in Copenhagen that recently recovered DNA from a piece of ancient chewing gum.

“Suddenly you’ve got these new techniques available and you take them and apply them to a new — well, old — mysterious substance,” Mr. Macleod said. His team is now hoping to track down more samples of the coveted material for scientific use.

While the research helps confirm that sperm whales, and possibly related species, too, are the source of the ambergris found along the world’s coasts, biologists still don’t know how this fancy fragrance really forms. When sperm whales were still being slaughtered, whalers found that only about one in a hundred yielded ambergris.

One leading theory, advanced in 2006, casts ambergris as what could generously be called a rectal pearl: Formed from layers of excrement that accumulated on an indigestible clump of squid beaks and worm cuticles. Mr. Macleod’s team, though, believes gut bacteria might play a bigger role in making it, a subject for additional study.

The long life span of ambergris and its potential to preserve DNA from long-dead whales could also help scientists estimate populations of sperm whales far before they were pushed to the brink of extinction. The genetic diversity of living sperm whales already suggests the species dwindled during past episodes of climate change, says Alana Alexander, who studies cetacean DNA at the University of Otago in New Zealand. But having samples from before the whaling era would help refine historical population estimates.

DNA might also be used to help prevent theft or illicit collection in places like Australia or the United States, where the substance is illegal under the Endangered Species Act.

Dr. Alexander’s only quibble is the current study’s sample size of ambergris, which she said is to be expected. “It’s certainly not within the average researcher’s project budget to pay wholesale price for it!”

Explore Nucleic Acid Extraction Protocols by Sample Type

The Technical Manual supplied with each kit contains recommended protocols for specific sample types. We continue to test additional sample types and publish the results as short Application Notes. Search our Application Notes database for your specific sample type of interest to find protocols for manual, Maxwell or plate-based methods.

Filter By

Obtaining good yields of high-quality DNA is a fundamental step critical to the success of many molecular biology applications.

Genomic DNA extraction methods isolate genomic DNA away from proteins, RNA and other cellular material. The basic steps involved in DNA isolation are: 1) Disruption of the cell structure to create a lysate 2) Protection of DNA from degradation during processing 3) Separation of the soluble DNA from cell debris and other insoluble material and 4) Elution of purified DNA.

Solution-based methods for DNA purification rely on precipitation and centrifugation steps to separate the genomic DNA in the cell lysate from other cellular materials. These methods use either organic extraction or &ldquosalting out&rdquo to separate soluble DNA from cellular proteins. Finally, the DNA is isolated by ethanol precipitation.

Solid-phase extraction methods involve binding of DNA to a solid support, such as silica or cellulose matrices, followed by washing and elution of the DNA from the solid support. Such methods can involve centrifugation, vacuum or magnetic methods to separate the bound DNA from other cellular components.

The best DNA extraction method to choose for any given situation will vary depending on your sample type, the number of samples you need to process, and the downstream application you are performing.

Monitoring endangered freshwater biodiversity using environmental DNA

Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data-driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals--representing amphibians, fish, mammals, insects and crustaceans--can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high-throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.

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Mahuku GS, Jara C, Cajiao C, and Beebe S (2003) Sources of resistance to angular leaf spot (Phaeoisariopsis griseola) in common bean core collection, wildPhaseolus vulgaris and secondary gene pool. Euphytica 130: 303–13.

Mahuku GS, Henríquez MA, Muñoz J, and Buruchara RA (2002) Molecular markers dispute the existence of the Afro-Andean group of the bean angular leaf spot pathogen,Phaeoisariopsis griseola. Phytopathology 92: 580–9.

Michaels SD, John MC, and Amasino RM (1994) Removal of polysaccharides from plant DNA by ethanol precipitation. Biotechniques 17: 274–6.

Miklas PN, Afanador L, and Kelly JD (1996) Recombination-facilitated RAPD marker-assisted selection for disease resistance in common bean. Crop Sci 36: 86–90.

Milgroom MG and Fry WE (1997) Contributions of population genetics to plant disease epidemiology and management. Adv Bot Res 24: 1–30.

Mitchell JL, Roberts PJ, and Moss ST (1995) Sequence or structure? A short review on the application of nucleic acid sequence information to fungal taxonomy. Mycologist 9: 67–75.

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More women than men have added their DNA to the human gene pool

More women than men have contributed to the gene pool of humanity since the first modern humans walked out of Africa around 70,000 years ago, according to a study.

Though the laws of biology state that male and female DNA should contribute roughly equally to the next generation, they are silent on how many of each sex must be involved in the business.

Researchers in Germany found that throughout human history, mothers have regularly outnumbered fathers, meaning that more women have passed on their DNA than men.

There might have been more women than men around in the past, an imbalance that could help to explain the results. But the researchers point to cultural biases, whereby relatively few men got to mate with multiple women and women tended to move home to live with their partners.

“Imagine a population of 100 females and 100 males,” said Mark Stoneking, who led the study at the Max Planck Institute for Evolutionary Anthropology. “If all the females but only one of the males reproduced, then while the males and females contribute 50:50 to the next generation, the male contribution is all from just one male.” The next generation would all have the same Y chromosome but 100 different sets of mitochondrial DNA, which is passed solely down the maternal line.

Stoneking’s team gathered together the genomes of 623 men from 51 populations around the world. They then compared the genetic diversity of the male Y chromosomes with the diversity of the men’s mitochondrial DNA.

They found that genetic differences between human populations were almost always larger for the Y chromosome than for mitochondrial DNA. The only exception was East Asia.

Using computer models, the researchers showed that the differences in genetic diversity arose if more women than men were breeding throughout human history. According to their simulation, an ancestral population of 60 women and 30 men were breeding in Africa before humans left the continent. The numbers fell to around 25 women and 15 men breeding at the time of the first migration of Homo sapiens, around 70,000 years ago. The whole population would have been larger, but the extras were not contributing to the gene pool.

As modern humans moved into Europe more than 45,000 years ago, the number of mothers may have outnumbered fathers by around 100 to 30, according to Stoneking. His study appears in the journal, Investigative Genetics.

In static populations, genetic diversity falls over time because some people do not have children, so their genetic quirks die out. But the tradition of women moving to be with their partners helped to counter the genetic decline by importing fresh DNA.

“What we’ve found is that there are significant differences in the history of human males and females in different parts of the world. Understanding why that’s the case and what are the social historical processes that led to those differences are what we want to investigate now,” said Stoneking.

The First Humans in Asia: A Genetic Mapping

Why did ancestries that persisted for so long vanish from the gene pool of people alive now? Ancient farmers carry the key to that answer.

The very first human beings originally emerged in Africa before spreading across Eurasia about 60,000 years ago. After that, the story of humankind heads down many different paths, some more well-studied than others.

Eastern regions of Eurasia are home to approximately 2.3 billion people today – roughly 30% of the world’s population. Archaeologists know from fossils and artifacts that modern humans have occupied Southeast Asia for 60,000 years and East Asia for 40,000 years.

But there’s a lot left to untangle. Who were the people who first came to these regions and eventually developed agriculture? Where did different populations come from? Which groups ended up predominant and which died out?

Ancient DNA is helping to answer some of these questions. By sequencing the genomes of people who lived many millennia ago, scientists like me are starting to fill in the picture of how Asia was populated.

Analyzing Ancient Genomes

In 2016, I joined Dr. Qiaomei Fu’s Molecular Paleontology Lab at the Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences in Beijing. Our challenge: Resolve the history of humans in East Asia, with the help of collaborators who were long dead – ancient humans who lived up to tens of thousands of years ago in the region.

Members of the lab extracted and sequenced ancient DNA using human remains from archaeological sites. Then Dr. Fu and I used computational genomic tools to assess how their DNA related to that of previously sequenced ancient and present-day humans.

One of our sequences came from ancient DNA extracted from the leg bones of the Tianyuan Man, a 40,000-year-old individual discovered near a famous paleoanthropological site in western Beijing. One of the earliest modern humans found in East Asia, his genetic sequence marks him as an early ancestor of today’s Asians and Native Americans. That he lived where China’s current capital stands indicates that the ancestors of today’s Asians began placing roots in East Asia as early as 40,000 years ago.

Farther south, two 8,000- to 4,000-year-old Southeast Asian hunter-gatherers from Laos and Malaysia associated with the Hòabìnhian culture have DNA that, like the Tianyuan Man, shows they’re early ancestors of Asians and Native Americans. These two came from a completely different lineage than the Tianyuan Man, which suggested that many genetically distinct populations occupied Asia in the past.

But no humans today share the same genetic makeup as either Hòabìnhians or the Tianyuan Man, in both East and Southeast Asia. Why did ancestries that persisted for so long vanish from the gene pool of people alive now? Ancient farmers carry the key to that answer.

DNA Carries Marks of Ancient Migrations

Based on plant remains found at archaeological sites, scientists know that people domesticated millet in northern China’s Yellow River region about 10,000 years ago. Around the same time, people in southern China’s Yangtze River region domesticated rice.

Unlike in Europe, plant domestication began locally and was not introduced from elsewhere. The process took thousands of years, and societies in East Asia grew increasingly complex, with the rise of the first dynasties around 4,000 years ago.

That’s also when rice cultivation appears to have spread from its origins to areas farther south, including lands that are today’s Southeast Asian countries. DNA helps tell the story. When rice farmers from southern China expanded southward, they introduced not only their farming technology but also their genetics to local populations of Southeast Asian hunter-gatherers.

The overpowering influx of their DNA ended up swamping the local gene pool. Today, little trace of hunter-gatherer ancestry remains in the genes of people who live in Southeast Asia.

Farther north, a similar story played out. Ancient Siberian hunter-gatherers show little relationship with East Asians today, but later Siberian farmers are closely related to today’s East Asians. Farmers from northern China moved northward into Siberia bringing their DNA with them, leading to a sharp decrease in prevalence of the previous local hunter-gatherer ancestry.

Past Populations were More Diverse than Today’s

Genetically speaking, today’s East Asians are not very different from each other. A lot of DNA is needed to start genetically distinguishing between people with different cultural histories.

What surprised Dr. Fu and me was how different the DNA of various ancient populations were in China. We and others found shared DNA across the Yellow River region, a place important to the development of Chinese civilization. This shared DNA represents a northern East Asian ancestry, distinct from a southern East Asian ancestry we found in coastal southern China.

When we analyzed the DNA of people who lived in coastal southern China 9,000-8,500 years ago, we realized that already by then much of China shared a common heritage. Because their archaeology and morphology was different from that of the Yellow River farmers, we had thought these coastal people might come from a lineage not closely related to those first agricultural East Asians. Maybe this group’s ancestry would be similar to the Tianyuan Man or Hòabìnhians.

But instead, every person we sampled was closely related to present-day East Asians. That means that by 9,000 years ago, DNA common to all present-day East Asians was widespread across China.

Today’s northern and southern Chinese populations share more in common with ancient Yellow River populations than with ancient coastal southern Chinese. Thus, early Yellow River farmers migrated both north and south, contributing to the gene pool of humans across East and Southeast Asia.

The coastal southern Chinese ancestry did not vanish, though. It persisted in small amounts and did increase in northern China’s Yellow River region over time. The influence of ancient southern East Asians is low on the mainland, but they had a huge impact elsewhere. On islands spanning from the Taiwan Strait to Polynesia live the Austronesians, best known for their seafaring. They possess the highest amount of southern East Asian ancestry today, highlighting their ancestry’s roots in coastal southern China.

Other emerging genetic patterns show connections between Tibetans and ancient individuals from Mongolia and northern China, raising questions about the peopling of the Tibetan Plateau.

Ancient DNA reveals rapid shifts in ancestry over the last 10,000 years across Asia, likely due to migration and cultural exchange. Until more ancient human DNA is retrieved, scientists can only speculate as to exactly who, genetically speaking, lived in East Asia prior to that.

This article is republished from The Conversation under a Creative Commons license. Read the original article.

Medieval DNA suggests Columbus didn’t trigger syphilis epidemic in Europe

In the late 1400s, a terrifying disease erupted in Europe, leaving victims with bursting boils and rotting flesh. The syphilis epidemic raged across the continent, killing up to 5 million people. For centuries, historians, and archaeologists have debated the origin of the disease, with some blaming Christopher Columbus and his crew for bringing it back from the Americas. Now, using DNA of the pathogen extracted from the remains of nine Europeans, researchers have found evidence that the epidemic was homegrown: Diverse syphilis strains were circulating in Europe, perhaps decades before Columbus’s voyages.

Today, syphilis and other conditions caused by the same bacterium, Treponema pallidum, such as yaws and bejel, are making a comeback, with millions of people infected every year. “These diseases are not just a problem of the past,” says Verena Schuenemann, a paleogeneticist at the University of Zürich and co-author of the new study. By understanding when and where T. pallidum originated, and how it has evolved, she says, researchers can learn how it might behave in the future and be prepared to treat it.

Researchers have long clashed over the circumstances of the 1495 European syphilis epidemic. The so-called Columbian theory posits that Columbus and his crew carried the bacterium, or an earlier progenitor of it, when they returned to Europe in 1493 after their American journey. Skeletons of Native Americans who died prior to Columbus’s arrival show bone lesions from Treponemal diseases, including yaws and bejel, and some researchers suspect syphilis was also present. However, other researchers believe syphilis itself circulated in Europe for centuries and became more virulent in the late 1400s. They point to a growing body of archaeological evidence: skeletal remains from across Europe with suggestive bone lesions, some possibly dating to the 14th century. Yet the evidence has always been inconclusive: Bone lesions can be caused by any of the Treponemal diseases, and some people with syphilis may not develop skeletal signs.

Now, a team of scientists has examined nine skeletons with suspected syphilis from five archaeological sites in Finland, Estonia, and the Netherlands. The researchers ground the bones into powder and analyzed it for signs of Treponemal DNA, which is notoriously difficult to recover because the bacterium is present only in small amounts and decomposes quickly. “Five years ago, everybody would have said it was impossible,” says co-author Johannes Krause, an archaeogeneticist and co-director of the Max Planck Institute for the Science of Human History.

Researchers reconstructed pathogen genomes from bones with signs of Treponemal disease.

The researchers managed to recover and sequence Treponemal DNA from four samples and compared the sequences with a modern syphilis strain. They used a molecular clock technique that tracks changes in the genes over time to estimate the ages of the strains, and calibrated those ages with carbon dating of the skeletons and wood of the coffins they were buried in.

The team went looking for syphilis, but what they found was a much wider array of Treponemal strains: not just syphilis, but also yaws, which today is found exclusively in the tropics, and a previously undiscovered strain with no modern-day counterpart. “We see that many different lineages were present in Europe, which we did not know before,” Schuenemann says. What’s more, the dating range given to two strains is bounded on the lower end by ages in the early to mid-1400s—potentially the first DNA evidence that syphilis existed in Europe prior to Columbus’s contact with the Americas, the team reports today in Current Biology .

Although the radiocarbon dates are inherently uncertain and are bounded at the upper end by dates into the early 1600s, the diversity of strains around the time of Columbus’s crossing offers additional evidence that the pathogen had already made a home in Europe. Diversity takes time to evolve, Krause says: “Either Columbus brought a whole bouquet of strains, or this diversity was present there before.”

Molly Zuckerman, a bioarchaeologist at Mississippi State University who studies ancient Treponemal disease, praises the researchers’ feat of extracting Treponemal DNA, but notes that the sample date ranges are wide and can’t fully disprove the Columbus hypothesis. “This paper does not provide that kind of golden prize of evidence of syphilis in the pre-Columbian period in the Old World.”

Evolutionary epidemiologist Edward Holmes of the University of Sydney agrees: “It’s really interesting and really important that they’ve got these syphilis strains at around that time. What I’m less sure about is the exact time scale of the samples.”

Krause admits he could use more European samples, dated more precisely to the pre-Columbian period. “It’s not yet the final nail in the coffin,” he says. The next step is to screen more skeletal material for older DNA from both the Old and New World, and nail down exactly which T. pallidum strains were present in each before Columbus made contact.

DNA Analysis Reveals Common Origin of Tianyuan Humans and Native Americans, Asians

An international team of scientists has sequenced nuclear and mitochondrial DNA extracted from remains of a 40,000-year-old human found at the Tianyuan Cave site near Beijing, China. The results show Tianyuan humans shared a common origin with ancestors of many present-day Asians and Native Americans.

Tree of the Tianyuan and 36 present-day mtDNAs, numbers indicate individuals in the tree and the map (Qiaomei Fu et al)

Humans with morphology similar to present-day humans appear in the fossil record across Eurasia between 40,000 and 50,000 years ago. The genetic relationships between these early modern humans and present-day human populations had not yet been established.

The team, including Dr Svante Pääbo and Dr Qiaomei Fu from the Max Planck Institute for Evolutionary Anthropology, extracted nuclear and mitochondrial DNA from a leg bone found in 2003 at the Tianyuan Cave site, located outside Beijing.

For their study, described in the Proceedings of the National Academy of Sciences, the scientists used new techniques that can identify ancient genetic material from an archaeological find even when large quantities of DNA from soil bacteria are present. They then reconstructed a genetic profile of the leg’s owner.

“This individual lived during an important evolutionary transition when early modern humans, who shared certain features with earlier forms such as Neanderthals, were replacing Neanderthals and Denisovans, who later became extinct,” Dr Pääbo said.

Researchers in the Tianyuan Cave (Institute of Vertebrate Paleontology and Paleoanthropology)

The findings reveal that the Tianyuan human related to the ancestors of many present-day Asians and Native Americans, but had already diverged genetically from the ancestors of present-day Europeans. In addition, this early modern human did not carry a larger proportion of Neanderthal or Denisovan DNA than present-day people in the region.

“More analyses of additional early modern humans across Eurasia will further refine our understanding of when and how modern humans spread across Europe and Asia,” Dr Pääbo concluded.

Bibliographic information: Qiaomei Fu et al. DNA analysis of an early modern human from Tianyuan Cave, China. PNAS, published online before print January 22, 2013 doi: 10.1073/pnas.1221359110

Watch the video: 3. DNA Extraction for CALeDNA (June 2022).


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