Category Archives: DNA

Lawrence Berkeley National Laboratory has made available for licensing a DNA extraction and isolation method that its inventors claim is more efficient, sensitive, and selective than current commercial DNA extraction kits.

In particular, the new technique may be especially valuable for downstream applications where the extraction of minute amounts of DNA plays a critical role, such as basic and applied biology research, forensics, biosecurity, and environmental testing, according to the technology's inventors.

"This is a general method to get DNA out from any kind of sample, but with higher sensitivity, we think, than current methods, and more versatility in terms of product models," Youn-Hi Woo, a staff scientist at Berkeley Lab and one of the method's inventors, told PCR Insider this week.

"It has a very broad field of use anywhere someone wants a very small amount of specific DNA from larger samples or a larger pool," she added.

According to the LBNL researchers, the most popular current commercial DNA extraction methods use detergents, paramagnetic particles, or membrane filters. Each of these methods works well for certain applications, but each also has drawbacks, such as non-specific DNA separation and contamination with salts or negatively charged polymers. In almost all cases, the various methods require that researchers perform extra time-consuming or laborious wash steps.

Furthermore, although paramagnetic particles eliminate many of the chemistry-related problems, they are difficult to employ using large sample volumes, meaning that researchers must first concentrate a sample down to microliter-scale volumes or less. This is particularly daunting with the larger-volume samples commonly found in environmental testing or forensics.

The new DNA extraction protocol, which the LBNL researchers described in a paper published earlier this year in Analytical Biochemistry, relies on the combination of the DNA-specific enzyme methyltransferase, or DNA Mtase, and so-called "click" chemistry, which has the ability to irreversibly couple two molecules under mild conditions.

More specifically, DNA in a complex sample is selectively labeled using MTaqI, an Mtase derived from Thermus aquaticus, and with alkynyl-SAM, a cofactor molecule that supplies methyl groups that the MTaqI transfers to the DNA when it recognizes short nucleotide sequences.

Then, the mixture is applied to an azide-modified click chemistry surface in the presence of copper ions, where the selected DNA molecules become covalently bound. Standard or vigorous washing steps wash away any contaminants, leaving behind only the desired DNA molecules bound to the modified surface.

"One strength of this technology is pulling out DNA by covalent bonds," Woo said. "This means it can't be pulled off easily. You can be pretty harsh in the washing steps to get rid of whatever the DNA was contaminated with. This gives you [more] freedom in what you do to purify your sample."

Originally posted here:

LBNL Seeks Licensees for Highly Specific and Sensitive DNA Extraction Method

ENCODE (Image: Ed Yong)

The recent dustup over the ENCODE project and its confusing finding that 80% of DNA is functional surprises me greatly. What surprises me especially is that people are surprised by junk DNA. Unfortunately this time the scientists are also culpable since, while the publicity surrounding ENCODE has been a media disaster, the 80% claim originated in the scientific papers themselves. There is no doubt that the project itself which represents a triumph of teamwork, dogged pursuit, technological mastery and first-rate science has produced enormously useful data, and there is no doubt it will continue to do so. What is in doubt is how long it will take for the public damage to be repaired.

Theres a lot written about the various misleading statements about the project made by both scientists and journalists and I cannot add much to it. All I can do is to point to some excellent articles:Larry Moran has waged a longstanding effort to spread the true wisdom about junk DNA for years on his blog. Ed Yong exhaustively summarizes a long list of opinions, links and analysis. T. Ryan Gregory has some great posts dispelling the myth of the myth of junk DNA. And John Timmer has the best popular account of the matter. The biggest mistake on the part of the scientists was to define functional so loosely that it could mean pretty much all of DNA. The second big mistake was not in clarifying what functional means to the public.

But what I found astonishing was why its so hard for people to accept that much of DNA must indeed be junk. Even to someone like me who is not an expert, the existence of junk DNA appeared perfectly normal. I think that junk DNA shouldnt shock us at all if we accept the standard evolutionary picture.

The standard evolutionary picture tells us that evolution is messy, incomplete and inefficient. DNA consists of many kinds of sequences. Some sequences have a bonafide biological function in that they are transcribed and then translated into proteins that have a clear physiological role. Then there are sequences which are only transcribed into RNA which doesnt do anything. There are also sequences which are only bound by DNA-binding proteins (which was one of the definitions of functional the ENCODE scientists subscribed to). Finally, there are sequences which dont do anything at all. Many of these sequences consist of pseudogenes and transposons and are defective and dysfunctional genes from viruses and other genetic flotsam, inserted into our genome through our long, imperfect and promiscuous genetic history. If we can appreciate that evolution is a flawed, piecemeal, inefficient and patchwork process, we should not be surprised to find this diversity of sequences with varying degrees of function or with no function in our genome.

The reason why most of these useless pieces have not been weeded out is simply because there was no need to. We should remember that evolution does not work toward a best possible outcome, it can only do the best with what it already has. Its too much of a risk and too much work to get rid of all these defective and non-functional sequences if they arent a burden; the work of simply duplicating these sequences is much lesser than that of getting rid of them. Thus the sequences hung around in our long evolutionary history and got passed on. The fact that they may not serve any function at all would be perfectively consistent with a haphazard natural mechanism depending on chance and the tacking on of non-functionality to useful functions simply as extra baggage.

There are two other facts in my view which should make it very easy for us to accept the existence of junk DNA. Consider that the salamander genome is ten times the size of the human genome. Now this implies two possibilities; either salamanders have ten times functional DNA than we do, or that the main difference between us and salamanders is that they have much more junk DNA. Wouldnt the complexity of salamander anatomy of physiology be vastly different if they really had so much more functional DNA? On the contrary, wouldnt the relative simplicity of salamanders compared to humans be much more consistent with just varying degrees of junk DNA? Which explanation sounds more plausible?

The third reason for accepting the reality of junk DNA is to simply think about mutational load. Our genomes, as of other organisms, have undergone lots of mutations during evolution. What would be the consequences if 90% of our genome were really functional and had undergone mutations? How would we have survived and flourished with such a high mutation rate? On the other hand, its much simpler to understand our survival if we assume that most mutations that happen in our genome happen in junk DNA.

As a summary then, we should be surprised to find someone who says they are surprised by junk DNA. Even someone like me who is not an expert can think of three reasons to like junk DNA:

1. The understanding that evolution is an inherently messy and inefficient process that often produces junk. This junk may be retained if its not causing trouble.

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Three reasons to like junk DNA

Researchers show that DNA supercoils are dynamic structures that can hop long distances, a phenomenon that could affect gene regulation.

Scientists understanding of how long strings of DNA are packaged into tiny spaces just got a little more complicated. New research on single molecules of DNA show that supercoilssegments of extra-twisted loops of DNAcan moving by jumping along a DNA strand. The results, published today (September 13) in Science, give researchers new insights into DNA organization and point to a surprisingly speedy mechanism of gene regulation inside cells.

This is the first study that addresses the dynamics of DNA supercoils, said Ralf Seidel, who studies movement of molecular motor proteins along DNA at the University of Technology Dresden, but was not involved in the research. This supercoil hopping motion allows DNA strands to transmit supercoiling, bringing sites together in very fast manner.

DNA, being a double helix, is naturally twisted. In vivo, its packaged with proteins called histones that help condense the millions or billions of nucleotides into the small space of a cells nucleus. Constant interaction with proteins moving along the strand, like transcription factors that need to open the helix to read the DNA sequence, can affect both the double helixs twist, and the strands writhethe coiling of the strand around itself. These extra-twisted coils, called plectonemes or supercoils, form not unlike coils in phone cords. By bringing together distant segments of DNA, such as regulatory elements and the genes they control, supercoiling can affect expression.

In order to get a better sense of how supercoils behave, Cees Dekker at Delft University of Technology and his colleagues induced supercoils in single strands of DNA molecules, labeled with fluorescent dye. One end of the DNA was anchored to the side of a glass capillary tube and a magnetic bead was attached to the other end. This allowed the researchers to use miniscule magnets to twist the DNA and induce supercoils, and watch their movement using fluorescence microscopy.

Unexpectedly, the team found that supercoils move along DNA strands in one of two ways. Sometimes they slowly diffuse along the strand; other times, the supercoils hoppeddisappearing suddenly from one location while simultaneously appearing at a distant location further down the strand.

This is far more complicated than diffusion of supercoils down the DNAs length, said Prashant Purohit, who studies DNA behavior at the University of Pennsylvania, but was not involved in the study. The DNA is behaving non-locally, he noted. It shows that writhethe coiling of the DNA strandis a global, not local quantity [of the strand].

So far the intriguing phenomenon has only been observed on single strands of naked DNA, Seidel cautioned, so its unclear how supercoils might act in vivo, when the DNA is well-packaged and studded with proteins. It may be that such behavior is more important for DNA in prokaryotic cells, which have less packaged DNA than eukaryotic cells, noted Bryan Daniels, who models biological systems at the Wisconsin Institutes for Discovery at the University of Wisconsin-Madison.

The ionic environment of the cell is also likely to influence supercoiling behavior. DNA is more likely to condense in the presence of multivalent ions (3 or more positive charges), for example, than in an environment of singly-valent ions. And Dekker and his colleagues, who used singly-valent ions in their experiments, found that more supercoils formed at lower concentrations of ions.

Dekker and his team are now looking at how different DNA sequences and the presence of DNA-binding proteins can influence supercoil formation and motionthe first step toward understanding supercoil movement in vivo.

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DNA with a Twist

A London familys DNA could be the missing link in a centuries-long quest to find the remains of King Richard III.

A team of archeologists at the University of Leicester in England exhumed a skeleton believed to be Richards beneath one of the universitys parking lots Wednesday and are hoping DNA evidence from the London family will prove their suspicions true.

Richard was killed in 1485 during the Battle of Bosworth often cited as the deciding battle in the War of the Roses by Henry Tudor VII, father of the famed King Henry VIII.

Richards Machiavellian rise to power its believed he had his nephews murdered in order to seize the thrown and short two-year reign as king is chronicled in Shakespeares play Richard III.

In 2005, British historian John Ashdown-Hill traced Richards bloodline to Joy Ibsen, a retired journalist who moved to London, Ont., from England after the Second World War and raised a family.

Ashdown-Hill discovered Ibsen and Richard shared a maternal ancestor, Cecily Neville.

Though Ibsen died in 2008, she passed the gene on to her three children: Michael, who lives in the U..K; Jeff, who lives in Toronto; and Leslie on Vancouver Island.

Its pretty exciting, said Jeff, 49. I wasnt expecting the findings to be so concise ... Im hoping that if theres a proper funeral for him, well get invited and maybe get a chance to rub elbows with some royals.

The skeleton exhumed Wednesday was found in whats believed to be the choir of the lost Church of the Grey Friars, the same place historical records indicate Richard was buried. Initial examinations found trauma to the skull consistent with a battle injury and a barbed arrow through the skeletons upper back.

Especially telling is the spinal deformity found on the exhumed skeleton. Its believed Richard had severe scoliosis, a form of spinal curvature that caused his right shoulder to appear higher than the left, the same type of curvature found on the skeleton.

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DNA could help ID a king

MADISON, Wis.--(BUSINESS WIRE)--

Forensic DNA professionals confront many challenges: cold case investigations, DNA backlogs and new applications like rapid DNA and kinship DNA testing. The 23rd International Symposium on Human Identification (ISHI) presents forensic professionals with an opportunity to learn about these and other developing forensic DNA technologies alongside fellow scientists, law enforcement professionals and forensic experts. This years ISHI will be held October 15-18 in Nashville, Tennessee at the Gaylord Opryland Resort.

As the largest conference on DNA analysis for human identification, the symposium attracts more than 800 DNA analysts and forensic scientists from around the world, providing these professionals an opportunity to explore and debate the latest research, technologies and ethical issues in the industry today. This years presenters and topics include:

Author and Educator Douglas Starr

Co-director of Boston Universitys graduate program in Science and Medical Journalism and author of Gold Dagger award-winning book The Little Killer of Shepherds: A True Crime Story and the Birth of Forensic Science, Starr is this years keynote speaker. In his latest book, Starr tells the story of forensic sciences 19th century pioneers and the notorious serial killer they caught and convicted using their new scientific techniques. Winner of the Gold Dagger award in the U.K. and a finalist for the Edgar Allen Poe award in the U.S., the book received laudatory reviews, including an Editors Choice listing in the New York Times Book Review and a place on the True Crime Bestseller lists of the Wall Street Journal and Library Journal.

SNA International Founder Amanda Sozer

SNA International lends expertise to forensic labs and mass fatality identification projects. Founder and President Amanda Sozer, who received recognition for her outstanding efforts during 9/11 and Hurricane Katrina, will be leading a workshop on forensic science and human rights at ISHI. The workshop will include speakers who have worked on human rights projects as well as a presentation on the AAAS Guidelines for Scientists and Human Rights Organizations, developed by a group of collaborating scientists and representatives of human right organizations. The guidelines are designed to be helpful to those establishing science and human rights partnerships and to facilitate and promote cooperation between scientists and human rights organizations seeking scientific expertise.

Sequencing the Black Death Genome: Hendrik Poinar

Hendrik Poinar and his colleagues at McMaster University in Hamilton, Ontario, Canada developed a technique to find and sequence the Black Death genome using the skeletal remains of its victims. The possibility of environmental contamination was high. To address this, Poinar and his team extracted the DNA using a molecular probe made from a modern strain of DNA, testing this new technique on approximately 100 samples of teeth and bone excavated from a London plague pit. The result was a strain of Y. pestis unlike any known today: the Black Death. Poinar will share details of this process during his talk at ISHI.

Workshops: DNA Backlog Reduction, Cold Case Investigative Techniques

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2012 International Symposium on Human Identification Features Emerging and Best Practice Forensic DNA Techniques ...

There was an extremely strong chance DNA found inside the underpants of a five-year-old girl came from the man accused of abusing her, a court has heard.

But the ACT Supreme Court has been told tests for saliva turned up nothing, despite the girls allegation her step-grandfather licked her vagina.

And the court has heard tests werent carried out on other items of clothing and bedding because they were likely to be covered in his DNA and have no probative value.

The underpants were also placed in the same bag as another item of clothing, prompting the defence to suggest the DNA might have transferred from one to the other.

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The 61-year-old man, who cannot be named, is on trial in front of Justice Richard Refshauge accused of two counts of having sexual intercourse with a child.

He has pleaded not guilty, and also denies two alternative charges of committing acts of indecency on the girl.

It is alleged he licked the girls vagina twice when he was babysitting her in April 2009.

The allegations came to light after the girls mother picked her up, when the girl asked her mother if she could tell her the secret she shared with poppy.

The accused man entered the witness box this afternoon and denied any wrongdoing, describing his shock when police confronted him with the allegations.

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Court hears DNA findings in child sex case

ScienceDaily (Sep. 11, 2012) DNA is constantly being damaged by environmental agents such as ultraviolet light or certain compounds present in cigarette smoke. Cells unceasingly implement repair mechanisms for this DNA, which are of redoubtable efficacy. A team from Institut Jacques Monod (CNRS/Universit Paris Diderot), in collaboration with scientists from the Universities of Bristol in the UK and Rockefeller in the USA, has for the first time managed to follow real-time the initial steps in one of these hitherto little known DNA repair systems. Working in a bacterial model, and thanks to an innovative technique applied to a single molecule of DNA, the scientists were able to understand how several actors interact to ensure the reliable repair of DNA.

Published in Nature on 9 September 2012, their work aims to better understand the onset of cancers and how they become resistant to chemotherapies.

Ultraviolet light, tobacco smoke or even the benzopyrenes contained in over-cooked meat can cause changes to the DNA in our cells, which may lead to the onset of cancers. These environmental agents deteriorate the actual structure of the DNA, notably causing so-called "bulky" lesions (like the formation of chemical bonds between DNA bases). In order to identify and repair this type of damage, the cell can call on several systems, such as transcription-coupled repair (TCR), whose complex mechanism of action still remains poorly understood today. Abnormalities affecting this TCR mechanism -- which permits permanent monitoring of the genome -- are the cause of some hereditary diseases such as Xeroderma pigmentosum, sufferers from which are hypersensitive to the Sun's ultraviolet rays and are commonly referred to as "children of the night."

For the first time, a team from Institut Jacques Monod (CNRS/Universit Paris Diderot), in collaboration with scientists at the Universities of Bristol in the UK and Rockefeller in the USA, has succeeded in observing the initial stages of TCR repair mechanisms in a bacterial model. To achieve this, they employed a novel technique for the nanomanipulation of individual molecules[1] which allowed them to detect and follow real-time the interactions between the molecules in play in a single damaged DNA molecule. They elucidated the interactions between different actors during the first steps of this TCR process. A first protein, RNA polymerase[2], usually crosses DNA without mishap, but is stalled when it meets a bulky lesion (like a train blocked on its rails by a landslide). A second protein, Mfd, binds to the stalled RNA polymerase and removes it from the damaged "rail" so that it can then replace it with the other proteins necessary to repair the damage. Measurements of the reaction speeds enabled the observation that Mfd acts particularly slowly on RNA polymerase, pushing it out of the way in about twenty seconds. Furthermore, Mfd does indeed displace stalled RNA polymerase, but then remains associated with the DNA for a longer period (of about five minutes), allowing it to coordinate the arrival of other repair proteins at the damaged site.

Although the scientists were able to explain how this system can achieve almost 100% reliability, a even clearer understanding of these repair processes is still essential in order to determine how cancers appear and subsequently may become resistant to chemotherapies.

Notes:

[1] During these nanomanipulation experiments, damaged DNA was grafted onto a glass surface on one side and a magnetic microbead on the other. The bead surface enabled the perpendicular extension of the DNA and measurement of this end-to-end extension using videomicroscopy. The binding to DNA of different proteins, and their action, is identifiable from the modification the protein generates in the structure or conformation of the DNA. This technique enables an extremely detailed structural and kinetic analysis of in vitro biochemical reactions.

[2] RNA polymerase is responsible for the reading of DNA by a gene and its rewriting in an RNA form, a process known as transcription. It has been shown that RNA polymerase does not only transcribe genes, but also the DNA between genes (until recently referred to as "junk" DNA), allowing, for example, polymerase RNA to perform its quality control by TCR on the entire genome of an organism.

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Real-time observation of single DNA molecule repair

The lexicon of science is riddled with catchy yet misleading terms. The god particle is nothing of the sort. Genes cannot really be selfish, and when astronomers talk about metals, they usually mean something else entirely. Now, we must add junk DNA to the list of scientific misnomers.

Last week, the results of the multinational Encode Project were published across 30 papers in the journals Nature, Science, Genome Biology and Genome Research. The five-year collaboration involved some 450 scientists working in 32 institutions and took up 300 years of computer time. The goal was to analyse the vast bulk of human DNA that does not constitute a gene ie, does not directly code for the creation of particular proteins and is seemingly surplus to requirements.

The conclusion? That this DNA is not junk at all, but absolutely vital for the functioning of our cells. It turns out that as much as a fifth of the 98 per cent of our DNA that falls into this category is instead made up, among other things, of switches bits of DNA that turn some genes on and others off. It is now believed that, in order to get to grips with genetic illnesses such as hereditary heart disease, some forms of diabetes and Crohns Disease, we need to understand these regulatory elements as much as the genes themselves.

It has been clear for a long time that there is a lot more to DNA than just genes. Indeed, one of the great scientific surprises in recent decades has been the discovery that the human genome is surprisingly bereft of actual genes. When the first draft of it was published in the summer of 2001, it did not describe the 100,000 or more genes that most biologists assumed we had, but fewer than 20,000 making Homo sapiens not much more well-endowed genetically than a fruit fly or even a lump of yeast. As an editorial in Nature put it, Unless the human genome contains a lot of genes that are opaque to our computers, it is clear we do not gain our undoubted complexity over worms and plants by using many more genes.

Partly as a result, the idea that scanning a persons genome can tell us pretty much everything about them their likely intelligence, the chance of criminal tendencies, their probable age and cause of death is now seen as a simplistic fantasy. Indeed, the more we learn about our genome, the more complex the story becomes. We have genes that tell our bodies to make proteins, genes that affect other genes, genes that are influenced by the environment, segments of DNA that switch certain genes on and off, as well as our RNA, the still-not-fully understood messenger molecule that conveys information from our DNA to protein factories in the cells.

Despite the fanfare with which the Encode findings were greeted last week, biologists have known for years that junk DNA, a term coined in 1972 by the Japanese-American geneticist Susumu Ohno, performs a host of functions, among them gene regulation. Indeed, it was always obvious that much of our DNA must be tasked with the activation or suppression of other parts of itself: genes that make bone tissue are present in all cells but are only switched on in bone cells; heart muscle genes are present but inactive in your teeth and liver and everywhere else.

Furthermore, as Ohno pointed out, a great deal of the genome consists of pseudogenes non-functioning copies of active genes that form the raw material of evolution. Without this spare genetic material, natural selection would have nothing to act upon. We have also known for some time that the dark part of our genome contains what are known as human endogenous retroviruses: bits of the genetic code from viruses that are a legacy of our long battle with these microbes. In millennia to come, it is likely that bits of the genome for HIV will become similarly incorporated into our DNA, as a legacy of the Aids epidemic.

The more we learn, the more the recipe book of life turns out to resemble less a single tome than a well-organised library, complete with a sophisticated index and with the ability to lend and borrow books. Some of the volumes are crucial a mix-up in the code could kill or cripple us while others moulder in the stacks. There is probably a lot of built-in redundancy, which is not surprising considering that the genomes of any species are the result of three billion years of evolution. Perhaps the most amazing thing is that we can make any sense of it at all.

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'Junk DNA' and the mystery of mankind's missing genes

A Massive Research Effort Now Shows How the Genome Works

By Daniel J. DeNoon WebMD Health News

Reviewed by Louise Chang, MD

Sept. 7, 2012 -- In what scientists call the biggest breakthrough in genetics since the unraveling of the human genome, a massive research effort now shows how the genome works.

The human genome contains 3 billion letters of code containing a person's complete genetic makeup.

The biggest surprise is that most of the DNA in the genome -- which had been called "junk DNA" because it didn't seem to do anything -- turns out to play a crucial role. While only 2% of the genome encodes actual genes, at least 80% of the genome contains millions of "switches" that not only turn genes on and off, but also tell them what to do and when to do it.

Eleven years ago, the Human Genome Project discovered the blueprint carried by every cell in the body. The new ENCODE project now has opened the toolbox each cell uses to follow its individual part of the blueprint. The effort is the work of more than 400 researchers who performed more than 1,600 experiments.

The genome, with its 3-billion-letter code, reads from beginning to end like a book. But in real life, the genome isn't read like a book. The ENCODE data shows it's an intricate dance, with each step carefully choreographed.

Ewan Birney, PhD, associate director of the European Molecular Biology Laboratory, was one of the leaders of the Human Genome Project. He also helped lead the ENCODE project.

"The ENCODE data is just amazing. It shows how complex the human genome is," Birney said at a news conference. "This is the science for this century. We are going to be working out how we make humans, starting out from a simple instruction manual."

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Genetics Breakthrough Changes Thinking About DNA

Last week, in response to a media blitz promoting a $288 million DNA project called ENCODE, headlines announced that most of our DNA formerly known as "junk" was actually useful.

A number of scientists both inside the study and out took issue with this claim - which centered on the 98 percent of our DNA that isn't officially part of any gene.

Sorting the workers from the freeloaders in our DNA is crucial to understanding how our genetic code works, how it drives human evolution and influences our traits and health.

Some biologists dislike the term "junk DNA" because they already knew at least part of it is doing something essential - like regulating how the instructions in the genes are carried out.

The genes hold recipes for making proteins - the working parts and scaffolding of the body. Some of the rest of the DNA tells the genes how much of a given protein to make at any given time.

The goal of the ENCODE (Encylopedia of DNA Elements) project was to figure out which parts have those important regulatory jobs.

According to some scientists involved, they succeeded in pinning down where many of those regulators lurked and identified variants in that DNA that other studies have connected to a variety of diseases. Those findings could lead to new targets for drug research and new avenues for predictive genetic testing.

But long before this project was conceived, scientists had begun to explore our jungle of mystery DNA. The question of non-gene DNA came up in 1975, when researchers discovered that humans and chimpanzees were 98 percent genetically identical. That meant we and chimps were more closely related than mice were to rats, or chimps were to gorillas.

The researchers who did the comparison pointed out that some of our differences might stem not from the genes, but from our other DNA that is regulating the genes.

That regulatory role is crucial when animals are developing in the womb. Some stretch of non-gene DNA could, for example, signal the human brain to keep growing long after chimp brain development would have shut off.

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Planet of the Apes: What is that big hunk of 'junk' DNA up to ?