Amid patent lawsuit, genetic sequencing upstart unveils new technology

Oxford Nanopore Technologies, the company that pioneered a new DNA analysis technique called nanopore sequencing, has remained secretive about the microscopic channel at the heart of its products. But on the heels of a patent infringement lawsuit from sequencing behemoth Illumina, Inc., the company has revealed what will drive its newest devices—a bacterial pore that seems to circumvent Illumina’s challenge.

Nanopore sequencing, hailed as a more portable and affordable way to analyze DNA than previous methods, works by measuring changes in electrical current as single nucleotides pass through a pore not much bigger than DNA itself. In 2014, Oxford Nanopore rolled out the first commercial nanopore sequencer, a handheld device called MinION. Although researchers have praised the technology and worked to improve its accuracy, they could only speculate about how MinION had succeeded where Illumina and others had failed; the structure of the MinION’s pore has remained a trade secret. In a lawsuit filed 23 February, Illumina suggested that Oxford Nanopore used a bacteria-derived pore called Mycobacterium smegmatis porin (Msp). Illumina licenses patents on an Msp system from the University of Washington, Seattle, and the University of Alabama, Birmingham, and claimed that Oxford Nanopore’s sequencers infringed those patents.

The suit came as distressing news to some researchers, who feared they might lose access to the firm’s nanopore products, or that the battle would stifle the development of new platforms. “I would love it if Illumina had released a platform. Instead what they’re doing is releasing a lawsuit,” says Winston Timp, a biomedical engineer at Johns Hopkins University in Baltimore, Maryland, whose lab has also licensed patents to Oxford Nanopore.

A webcast presentation today for customers of the company will likely put many of those fears to rest. Oxford Nanopore’s Chief Technology Officer Clive Brown announced an upgrade to its devices—a new pore the company has been describing in presentations as R9. Brown revealed that R9 is CsgG, a membrane protein derived from Escherichia coli, but present in many bacterial species.

CsgG is a relative newcomer to the lineup of nanopore candidates. A team led by Han Remaut, a molecular biologist at the Flanders Institute for Biotechnology in Belgium, worked out the structure of CsgG and published it in 2014. The pore seemed to hold promise for sequencing because it has a very narrow and well-defined passage for a DNA strand, Remaut says. Many other pores that have been explored, including Msp, taper to their narrowest points more gradually and over a longer distance, he explains. That allows for more interactions between the pore and a DNA strand, which can degrade the electrical signals of interest for sequencing.

CsgG seems to outperform its (still unidentified) predecessor, R7, at discriminating nucleotides as they pass through, Brown said. He showed preliminary results suggesting that where the R7 system ranged from 75% to 90% accuracy (depending on the how the DNA is read through the pore), the new system that includes R9 achieved 85% to 95%. Brown didn’t reveal anything about the identity of R7, but said the company will transition all its customers to R9 this spring. A more powerful sequencer known as PromethION, set to ship to researchers at the end of March, will only contain R9. Oxford Nanopore holds an exclusive license on the use of the pore, Brown said today, and has developed more than 700 variations of it. “It’s the way forward, as far as we’re concerned.”

Oxford Nanopore also responded directly to Illumina’s suit this week. In an 8 March letter to the U.S. International Trade Commission, the company denies all charges of infringement, and claims that Illumina is acting on “unsubstantiated speculation” to block Oxford Nanopore products and maintain its own monopoly over conventional DNA sequencing. Illumina has never proved that it’s capable of making its own working nanopore sequencer, the letter alleges, and removing Oxford Nanopore technology from the market as Illumina requests could harm public health research efforts such as the tracking of Ebola and Zika viruses in the field.


This article was originally published on Science.  Read the original article.

CRISPR: gene editing is just the beginning

Whenever a paper about CRISPR–Cas9 hits the press, the staff at Addgene quickly find out. The non-profit company is where study authors often deposit molecular tools that they used in their work, and where other scientists immediately turn to get them. It is also where other scientists immediately turn to get their hands on these reagents. “We get calls within minutes of a hot paper publishing,” says Joanne Kamens, executive director of the company in Cambridge, Massachusetts.


Addgene’s phones have been ringing a lot since early 2013, when researchers first reported1, 2, 3 that they had used the CRISPR–Cas9 system to slice the genome in human cells at sites of their choosing. “It was all hands on deck,” Kamens says. Since then, molecular biologists have rushed to adopt the technique, which can be used to alter the genome of almost any organism with unprecedented ease and finesse. Addgene has sent 60,000 CRISPR-related molecular tools — about 17% of its total shipments — to researchers in 83 countries, and the company’s CRISPR-related pages were viewed more than one million times in 2015.

Much of the conversation about CRISPR–Cas9 has revolved around its potential for treating disease or editing the genes of human embryos, but researchers say that the real revolution right now is in the lab. What CRISPR offers, and biologists desire, is specificity: the ability to target and study particular DNA sequences in the vast expanse of a genome. And editing DNA is just one trick that it can be used for. Scientists are hacking the tools so that they can send proteins to precise DNA targets to toggle genes on or off, and even engineer entire biological circuits — with the long-term goal of understanding cellular systems and disease.

“For the humble molecular biologist, it’s really an extraordinarily powerful way to understand how the genome works,” says Daniel Bauer, a haematologist at the Boston Children’s Hospital in Massachusetts. “It’s really opened the number of questions you can address,” adds Peggy Farnham, a molecular biologist at the University of Southern California, Los Angeles. “It’s just so fun.”

Here, Nature examines five ways in which CRISPR–Cas9 is changing how biologists can tinker with cells.


Broken scissors

There are two chief ingredients in the CRISPR–Cas9 system: a Cas9 enzyme that snips through DNA like a pair of molecular scissors, and a small RNA molecule that directs the scissors to a specific sequence of DNA to make the cut. The cell’s native DNA repair machinery generally mends the cut — but often makes mistakes.

That alone is a boon to scientists who want to disrupt a gene to learn about what it does. The genetic code is merciless: a minor error introduced during repair can completely alter the sequence of the protein it encodes, or halt its production altogether. As a result, scientists can study what happens to cells or organisms when the protein or gene is hobbled.

But there is also a different repair pathway that sometimes mends the cut according to a DNA template. If researchers provide the template, they can edit the genome with nearly any sequence they desire at nearly any site of their choosing.

In 2012, as laboratories were racing to demonstrate how well these gene-editing tools could cut human DNA, one team decided to take a different approach. “The first thing we did: we broke the scissors,” says Jonathan Weissman, a systems biologist at the University of California, San Francisco (UCSF).

Weissman learned about the approach from Stanley Qi, a synthetic biologist now at Stanford University in California, who mutated the Cas9 enzyme so that it still bound DNA at the site that matched its guide RNA, but no longer sliced it. Instead, the enzyme stalled there and blocked other proteins from transcribing that DNA into RNA. The hacked system allowed them to turn a gene off, but without altering the DNA sequence4.

The team then took its ‘dead’ Cas9 and tried something new: the researchers tethered it to part of another protein, one that activates gene expression. With a few other tweaks, they had built a way to turn genes on and off at will5.


Several labs have since published variations on this method; many more are racing to harness it for their research6 (see ‘Hacking CRISPR’). One popular application is to rapidly generate hundreds of different cell lines, each containing a different guide RNA that targets a particular gene. Martin Kampmann, another systems biologist at UCSF, hopes to screen such cells to learn whether flipping certain genes on or off affects the survival of neurons exposed to toxic protein aggregates — a mechanism that is thought to underlie several neurodegenerative conditions, including Alzheimer’s disease. Kampmann had been carrying out a similar screen with RNA interference (RNAi), a technique that also silences genes and can process lots of molecules at once, but which has its drawbacks. “RNAi is a shotgun with well-known off-target effects,” he says. “CRISPR is the scalpel that allows you to be more specific.”


Weissman and his colleagues, including UCSF systems biologist Wendell Lim, further tweaked the method so that it relied on a longer guide RNA, with motifs that bound to different proteins. This allowed them to activate or inhibit genes at three different sites all in one experiment7. Lim thinks that the system can handle up to five operations at once. The limit, he says, may be in how many guide RNAs and proteins can be stuffed into a cell. “Ultimately, it’s about payload.”

That combinatorial power has drawn Ron Weiss, a synthetic biologist at the Massachusetts Institute of Technology (MIT) in Cambridge, into the CRISPR–Cas9 frenzy. Weiss and his colleagues have also created multiple gene tweaks in a single experiment8, making it faster and easier to build complicated biological circuits that could, for example, convert a cell’s metabolic machinery into a biofuel factory. “The most important goal of synthetic biology is to be able to program complex behaviour via the creation of these sophisticated circuits,” he says.

CRISPR epigenetics

When geneticist Marianne Rots began her career, she wanted to unearth new medical cures. She studied gene therapy, which targets genes mutated in disease. But after a few years, she decided to change tack. “I reasoned that many more diseases are due to disturbed gene-expression profiles, not so much the single genetic mutations I had been focused on,” says Rots, at the University Medical Center Groningen in the Netherlands. The best way to control gene activity, she thought, was to adjust the epigenome, rather than the genome itself.


The epigenome is the constellation of chemical compounds tacked onto DNA and the DNA-packaging proteins called histones. These can govern access to DNA, opening it up or closing it off to the proteins needed for gene expression. The marks change over time: they are added and removed as an organism develops and its environment shifts.

In the past few years, millions of dollars have been poured intocataloguing these epigenetic marks in different human cells, and their patterns have been correlated with everything from brain activity to tumour growth. But without the ability to alter the marks at specific sites, researchers are unable to determine whether they cause biological changes. “The field has met a lot of resistance because we haven’t had the kinds of tools that geneticists have had, where they can go in and directly test the function of a gene,” says Jeremy Day, a neuroscientist at the University of Alabama at Birmingham.

CRISPR–Cas9 could turn things around. In April 2015, Charles Gersbach, a bioengineer at Duke University in Durham, North Carolina, and his colleagues published9 a system for adding acetyl groups — one type of epigenetic mark — to histones using the broken scissors to carry enzymes to specific spots in the genome.

The team found that adding acetyl groups to proteins that associate with DNA was enough to send the expression of targeted genes soaring, confirming that the system worked and that, at this location, the epigenetic marks had an effect. When he published the work, Gersbach deposited his enzyme with Addgene so that other research groups could use it — and they quickly did. Gersbach predicts that a wave of upcoming papers will show a synergistic effect when multiple epigenetic markers are manipulated at once.

The tools need to be refined. Dozens of enzymes can create or erase an epigenetic mark on DNA, and not all of them have been amenable to the broken-scissors approach. “It turned out to be harder than a lot of people were expecting,” says Gersbach. “You attach a lot of things to a dead Cas9 and they don’t happen to work.” Sometimes it is difficult to work out whether an unexpected result arose because a method did not work well, or because the epigenetic mark simply doesn’t matter in that particular cell or environment.


Rots has explored the function of epigenetic marks on cancer-related genes using older editing tools called zinc-finger proteins, and is now adopting CRISPR–Cas9. The new tools have democratized the field, she says, and that has already had a broad impact. People used to say that the correlations were coincidental, Rots says — that if you rewrite the epigenetics it will have no effect on gene expression. “But now that it’s not that difficult to test, a lot of people are joining the field.”

CRISPR code cracking

Epigenetic marks on DNA are not the only genomic code that is yet to be broken. More than 98% of the human genome does not code for proteins. But researchers think that a fair chunk of this DNA is doing something important, and they are adopting CRISPR–Cas9 to work out what that is.

Some of it codes for RNA molecules — such as microRNAs and long non-coding RNAs — that are thought to have functions apart from making proteins. Other sequences are ‘enhancers’ that amplify the expression of the genes under their command. Most of the DNA sequences linked to the risk of common diseases lie in regions of the genome that contain non-coding RNA and enhancers. But before CRISPR–Cas9, it was difficult for researchers to work out what those sequences do. “We didn’t have a good way to functionally annotate the non-coding genome,” says Bauer. “Now our experiments are much more sophisticated.”

Farnham and her colleagues are using CRISPR–Cas9 to delete enhancer regions that are found to be mutated in genomic studies of prostate and colon cancer. The results have sometimes surprised her. In one unpublished experiment, her team deleted an enhancer that was thought to be important, yet no gene within one million bases of it changed expression. “How we normally classify the strength of a regulatory element is not corresponding with what happens when you delete that element,” she says.

“I wish I had had this technology sooner. My postdoc would have been a lot shorter.”

More surprises may be in store as researchers harness CRISPR–Cas9 to probe large stretches of regulatory DNA. Groups led by geneticists David Gifford at MIT and Richard Sherwood at the Brigham and Women’s Hospital in Boston used the technique to create mutations across a 40,000-letter sequence, and then examined whether each change had an effect on the activity of a nearby gene that made a fluorescent protein10. The result was a map of DNA sequences that enhanced gene expression, including several that had not been predicted on the basis of gene regulatory features such as chromatin modifications.

Delving into this dark matter has its challenges, even with CRISPR–Cas9. The Cas9 enzyme will cut where the guide RNA tells it to, but only if a specific but common DNA sequence is present near the cut site. This poses little difficulty for researchers who want to silence a gene, because the key sequences almost always exist somewhere within it. But for those who want to make very specific changes to short, non-coding RNAs, the options can be limited. “We cannot take just any sequence,” says Reuven Agami, a researcher at the Netherlands Cancer Institute in Amsterdam.

Researchers are scouring the bacterial kingdom for relatives of the Cas9 enzyme that recognize different sequences. Last year, the lab of Feng Zhang, a bioengineer at the Broad Institute of MIT and Harvard in Cambridge, characterized a family of enzymes called Cpf1 that work similarly to Cas9 and could expand sequence options11. But Agami notes that few alternative enzymes found so far work as well as the most popular Cas9. In the future, he hopes to have a whole collection of enzymes that can be targeted to any site in the genome. “We’re not there yet,” he says.

CRISPR sees the light

Gersbach’s lab is using gene-editing tools as part of an effort to understand cell fate and how to manipulate it: the team hopes one day to grow tissues in a dish for drug screening and cell therapies. But CRISPR–Cas9’s effects are permanent, and Gersbach’s team needed to turn genes on and off transiently, and in very specific locations in the tissue. “Patterning a blood vessel demands a high degree of control,” he says.


Gersbach and his colleagues took their broken, modified scissors — the Cas9 that could now activate genes — and added proteins that are activated by blue light. The resulting system triggers gene expression when cells are exposed to the light, and stops it when the light is flicked off12. A group led by chemical biologist Moritoshi Sato of the University of Tokyo rigged a similar system13, and also made an active Cas9 that edited the genome only after it was hit with blue light14.

Others have achieved similar ends by combining CRISPR with a chemical switch. Lukas Dow, a cancer geneticist at Weill Cornell Medical College in New York City, wanted to mutate cancer-related genes in adult mice, to reproduce mutations that have been identified in human colorectal cancers. His team engineered a CRISPR–Cas9 system in which a dose of the compound doxycycline activates Cas9, allowing it to cut its targets15.

The tools are another step towards gaining fine control over genome editing. Gersbach’s team has not patterned its blood vessels just yet: for now, the researchers are working on making their light-inducible system more efficient. “It’s a first-generation tool,” says Gersbach.


Cancer researcher Wen Xue spent the first years of his postdoc career making a transgenic mouse that bore a mutation found in some human liver cancers. He slogged away, making the tools necessary for gene targeting, injecting them into embryonic stem cells and then trying to derive mice with the mutation. The cost: a year and US$20,000. “It was the rate-limiting step in studying disease genes,” he says.

A few years later, just as he was about to embark on another transgenic-mouse experiment, his mentor suggested that he give CRISPR–Cas9 a try. This time, Xue just ordered the tools, injected them into single-celled mouse embryos and, a few weeks later — voilá. “We had the mouse in one month,” says Xue. “I wish I had had this technology sooner. My postdoc would have been a lot shorter.”

Researchers who study everything from cancer to neurodegeneration are embracing CRISPR-Cas9 to create animal models of the diseases. It lets them engineer more animals, in more complex ways, and in a wider range of species. Xue, who now runs his own lab at the University of Massachusetts Medical School in Worcester, is systematically sifting through data from tumour genomes, using CRISPR–Cas9 to model the mutations in cells grown in culture and in animals.

Researchers are hoping to mix and match the new CRISPR–Cas9 tools to precisely manipulate the genome and epigenome in animal models. “The real power is going to be the integration of those systems,” says Dow. This may allow scientists to capture and understand some of the complexity of common human diseases.


Take tumours, which can bear dozens of mutations that potentially contribute to cancer development. “They’re probably not all important in terms of modelling a tumour,” says Dow. “But it’s very clear that you’re going to need two or three or four mutations to really model aggressive disease and get closer to modelling human cancer.” Introducing all of those mutations into a mouse the old-fashioned way would have been costly and time-consuming, he adds.

Bioengineer Patrick Hsu started his lab at the Salk Institute for Biological Studies in La Jolla, California, in 2015; he aims to use gene editing to model neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease in cell cultures and marmoset monkeys. That could recapitulate human behaviours and progression of disease more effectively than mouse models, but would have been unthinkably expensive and slow before CRISPR–Cas9.

Even as he designs experiments to genetically engineer his first CRISPR–Cas9 marmosets, Hsu is aware that this approach may be only a stepping stone to the next. “Technologies come and go. You can’t get married to one,” he says. “You need to always think about what biological problems need to be solved.”



This article was originally published on Nature.  Read the original article.

DNA Barcodes Could Streamline Search for New Drugs to Combat Cancer

A little more than a decade ago, researchers began adapting a familiar commercial concept to genomics: the barcode. Instead of the black, printed stripes of the Universal Product Codes (UPCs) that we see on everything from package deliveries to clothing tags, they used short, unique snippets of DNA to label cells. These biological “barcodes” enable scientists to distinguish one cell type from another, in much the same way that a supermarket scanner recognizes different brands of cereal.

DNA barcoding has already empowered single-cell analysis, including for nerve cells in the brain. Now, in a new NIH-supported study, DNA barcoding helps in the development of a new method that could greatly streamline an increasingly complex and labor-intensive process: screening for drugs to combat cancer.

The new method, reported recently in the journal Nature Biotechnology, is called PRISM, short for Profiling Relative Inhibition Simultaneously in Mixtures [1]. In their trial run of PRISM, the researchers uniquely barcoded more than 100 cancer cell lines. This allowed them to pool the cell lines and screen them all at the same time (instead of individually) against each of thousands of potential drug compounds, to see which, if any, of the barcoded cells they had the power to kill. This proof of concept suggests that PRISM, with further refinements, could help to accelerate cancer drug discovery and bring greater precision to the screening process.

This innovative research was led by the lab of Todd Golub of the Broad Institute of MIT and Harvard, Cambridge, MA. PRISM consists of two key components. The first is a library of uniquely barcoded cancer cell lines. Golub and colleagues produced the library by using a virus to insert a distinct DNA barcode, just 24 nucleotides long, stably into the genomes of each genetically distinct tumor cell line.

The second component is a barcode detection system. It scans tiny, color-coded beads that are preprogrammed to bind to a specific barcode, providing a tally of each cell line present in a mixture. A special stain allows researchers to quantify the barcodes attached to each colored bead based on the intensity of their fluorescence.

In the study, the researchers first pooled five barcoded cancer cell lines to see if the PRISM method worked as intended. They found that the detection system had outstanding sensitivity, detecting as few as 10 uniquely barcoded cells in a pool of 4,000! In the next key test, the researchers exposed a mixture of 25 barcoded lung cancer cell lines to approved cancer drugs. For each drug, sensitive cells were killed, and so those barcodes dropped out—but the resistant cells and their barcodes were retained. They found that the drug responses from the barcoded cells largely mirrored those reported previously for the 25 cell lines.

Now that PRISM had passed these initial tests, the researchers moved on to screen 102 cancer cell lines across 8,400 compounds—most with unknown effects on cancer. Those screens revealed 90 chemicals that selectively killed some cancer cell lines and not others. Golub and his team focused in detail on one of those chemicals called BRD-7880. By comparing the effects of this novel compound to those of others in the screen, the researchers figured out how BRD-7880 works and which types of cancer cells it is likely to kill. This shows how PRISM could be employed to discover novel drug candidates with remarkable ease and also elucidate quickly their mechanisms of action.

With PRISM, it’s now feasible to screen entire small molecule libraries across large panels of cancer cell lines. The approach might also be put to use for optimizing drug candidates or for discovering new uses for cancer drugs that have already been FDA approved. The researchers say they have now produced about 700 barcoded cancer cell lines, and this collection can be expanded to represent thousands of genetically distinct human cancers.

The more scientists have learned about the diversity of mutations that drive various forms of cancer, the clearer it has become that successfully combating these diseases will require a diverse arsenal of targeted approaches to therapy. While that remains a tremendous challenge, this new method could help to bring the goal of discovering many more precise and effective cancer treatments within better reach.


This article was originally published on NIH.  Read the original article.

New TSRI Study Shows HIV Structure in Unprecedented Detail

A new study from scientists at The Scripps Research Institute (TSRI) describes the high-resolution structure of the HIV protein responsible for recognition and infection of host cells.

The study, published today in the journal Science, is the first to show this HIV protein, known as the envelope (Env) trimer, in its natural or “native” form. The findings also include a detailed map of a vulnerable site at the base of this protein, as well as the binding site of an antibody that can neutralize HIV.

“This structure has been elusive because its fragility typically causes it to fall apart before it can be imaged,” said TSRI Associate Professor Andrew Ward, senior author of the study. “Now that we know what the native state looks like, the next step is to look at vaccine applications.”

Studying HIV’s Defenses

Imagine an airplane going in for a landing. Now imagine the airport runway is covered with heaps of barbed wire.

This is the kind of challenge human antibodies face when they attempt to neutralize HIV.

“The immune system can generate a response, but those responses can’t effectively hit the virus,” said Ward.

Ideally, antibodies would be able to target HIV’s Env trimer—three loosely connected proteins that stick out of the virus’s membrane and enable the virus to fuse with and infect host cells. This “fusion machinery” is also a valuable target because its structure is highly conserved, meaning the same vulnerabilities exist on many strains of the virus, and antibodies against these sites could be “broadly neutralizing.” Unfortunately, a “shield” of sugar molecules, called glycans, blocks many antibodies from reaching this region.

To develop a vaccine against HIV, researchers need a detailed map of these glycans to reveal the small holes in the shield where antibodies might penetrate and neutralize the underlying viral machinery.

The HIV trimer is notoriously unstable, however, making it hard for scientists to capture a good image. Partly due to this limitation, previous studies at TSRI and other institutions had shown only truncated trimers or high-resolution models of mutation-stabilized trimers. No one had a clear view of the trimer and its glycan defenses in their native form.

New Techniques Lead to Detailed Map

In the new study, the researchers employed cryo-electron microscopy (EM)—a 3D imaging technique that enables resolution of atomic-level details. TSRI maintains a state-of-the-art cryo-EM suite that includes a powerful Titan Krios cryo-electron microscope and a new generation of digital camera, the Gatan K2 Summit.

The researchers devised a strategy to extract and purify the fragile HIV Env trimer from its membrane environment and load it into the microscope for imaging. The process involved the use of an HIV broadly neutralizing antibody, PGT151, previously discovered in the lab of TSRI Professor Dennis Burton (also scientific director of the International AIDS Vaccine Initiative’s (IAVI) Neutralizing Antibody Center and the National Institutes of Health (NIH)-sponsored Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), both at TSRI).

The resulting images included a more-complete trimer structure than ever seen before. Researchers could see the complete fusion machinery, complex glycans and a vaccine target called the membrane proximal external region (MPER). The structures also demonstrated that the trimer is malleable and can subtly alter its shape. This shape-shifting is both part of its fusion machinery and a way to dodge neutralizing antibody responses.

The structure also includes a highly detailed picture of the PGT151 site of vulnerability, the most complex and extensive broadly neutralizing epitope (site that antibodies can recognize) yet described. In addition to targeting several glycans on the surface of Env, PGT151 binds to the fusion peptide—rendering the virus unable to infect host cells.

In addition, the researchers used this more complete trimer to study an antibody that binds to MPER. In the past, 3D structures of this region had only been studied using trimer fragments.

The findings give researchers a better idea of the antibody traits needed to negotiate the glycan shield. “That’s extremely important to know when you’re trying to develop a vaccine against HIV,” said Jeong Hyun Lee, a graduate student in the Ward lab and first author of the study.

Ward said the newly solved structure is similar to the Env trimer-mimicking structures being developed for an HIV vaccine and confirms that vaccine strategies are on target. Researchers can now build on that work to develop superior vaccine candidates.


This article was originally published on The Scripps Research Institute.  Read the original article.

Viruses Have Their Own Version of CRISPR

With all the buzz around CRISPR, the gene-editing technique that has instigatedmany an ethical debate and one acrimonious patent dispute, it would be easy to mistake it for a recent human invention. It’s not. Bacteria invented CRISPR billions of years ago, as a defense against marauding viruses. The bacteria grab pieces of a virus’s genetic material and incorporate these fragments into their own DNA. In doing so, they memorize the identities of past enemies. They can then use the viral sequences to guide their own defensive enzymes.

Now, it seems that some viruses use the same trick. Bernard La Scola and Didier Raoult from Aix-Marseille University have found that some giant viruses have a CRISPR-esque immune system, which they use to defend themselves from other smaller viruses. It seems that the defenders steal the genes of the attackers, and use those ‘memorized’ sequences to tailor their own countermeasures.

This is the latest in a string of discoveries that show how unexpectedly crazy the viral world really is. In the last 13 years, scientists have found giant viruses that outsize bacteria, viruses that parasitize other viruses, and now viruses with immune systems that defend themselves against more viruses. Arms races, it seems, have truly gone viral.  

The story began in 1992, when La Scola and Raoult studied amoebas contaminating the water of a cooling tower in Bradford, England. The amoebas were infected by a microbe, which was so large that the researchers initially assumed it was a bacterium. Only later, in 2003, did they realize it was a virus—a huge one, around four times bigger than, say, HIV or the influenza virus. They called it mimivirus.

An entire world of giant viruses soon came to light: Mamavirus in a Parisian cooling tower, Pithovirus in 30,000-year-old Russian ice, and Megavirus andPandoravirus in Chilean coastal waters. Most of these also infect amoebas, manufacturing new copies of themselves by setting up viral factories in their hosts. And these factories can themselves be corrupted by viruses.

In 2008, La Scola and Raoult noticed that amoebas infected by Mamavirus often carry a second smaller virus. This pipsqueak is a parasite that hijacks Mamavirus’s factories, using them to make copies of itself at the expense of its bigger cousin. When it’s around, the giant virus reproduces slowly, assembles abnormally, and produces daughters that are poorer at infecting amoebas. The team described the smaller virus as a ‘virophage’—an ‘eater of viruses’, a virus that sickens other viruses.

That first virophage was called Sputnik, after the Russian for ‘fellow traveler.’ More were then discovered, including Maverick virus from coastal waters,Organic Lake virophage from an Antarctic lake, and Sputnik 2 found in theinflamed eye of a French teenager.

The latest member of the virophage club is Zamilon, after the Arabic for “neighbor.” Raoult and La Scola found in 2014, and they noticed that it can only parasitize some branches of the Mimivirus family tree. Of the three such branches, one—lineage A—is immune to Zamilon.

Raoult suggested that these giant viruses defend themselves from Zamilon with some kind of CRISPR-like immune system. In other words, they contain stolen copies of Zamilon’s DNA, and using these pilfered sequences, they deploy DNA-slicing enzymes to disable the virophages. “Bernard disagreed, so we competed among ourselves to find the answer,” says Raoult, laughing.

He made three predictions. First, the A-group mimiviruses should contain DNA that matched the Zamilon virophages (which they resist), but not the Sputnik ones (which they don’t). Second, these immunizing sequences would be absent in the other two Mimivirus lineages that were not resistant to Zamilon. Third, the stolen Zamilon sequences would be accompanied by enzymes for unwinding and cutting DNA. All three predictions were true. “The war between giant viruses and virophages is similar to that between bacteria and viruses,” says Raoult.

The giant virus’s defense system, which the team calls MIMIVRE, isn’t exactly the same as CRISPR, but it is very close in form. It’s a wonderful example of convergent evolution, where two groups of living things independently come up with the same solutions to the same problems.

“[Some giant viruses] can get sick from a viral infection and can produce a “immune” response to the infection,” says Chantal Abergel, who has herself discovered several giant viruses and virophages. “Again, this blurs the frontier between viruses and cells and ask for reconsideration of what should be considered as alive.”

Many scientists insist that viruses aren’t alive: They have no autonomy, they don’t metabolize, and they depend on other (living) organisms to reproduce. In the words of one researcher, they exist “on the border between chemistry and life.” Still, the question is far from settled: A recent poll by the Virology Blog, which asked readers about the status of viruses, arrived at an almost perfect three-way split between alive, not alive, and “something in between.”

It’s clear where Raoult stands. “Giant viruses are not ordinary viruses,” he says. He thinks of them just another type of microbe, a group of microscopic living organisms, much like bacteria. They have their own immune system—MIMIVIRE. And they have their own parasites—virophages. Even their parasites have parasites.

In 2012, Raoult’s team found that both Lentille virus (a giant virus) and Sputnik 2 (the virophage that parasitizes it) are parasitized by a selfish piece of jumping DNA. This sequence, which the team called a ‘transpoviron’ can hop in and out of the genomes of both viruses and make new copies of itself. Similar bits of mobile DNA exist within our own cells and those of other living things—another indicator of the blurred boundaries that these giant viruses straddle.

And we’ve only known about this hidden world of giant viruses, virophages, and transpovirons for just over a dozen years. As Raoult once said to me: “If you want to see something really bizarre, you have to look where you didn’t know to look in the first place.”



This article was originally published on The Atlantic.  Read the original article.

How much ‘junk’ is in our DNA?

Only a small fraction of our DNA contains genes that encode the proteins that go on to build who we are. So why do we have the rest of our genome?

Over many decades, the moniker “junk” has been broadly used to refer to non-coding sequences in our DNA that appear to lack any function. It was first used in the 1960s to suggest that the majority of our DNA may be expendable. The term “junk DNA” has become very popular, although it has deterred some from studying it. Who would seriously apply for funding to investigate junk?

Controversy erupts

In 2001, the first sequenced human genome surprised us all by identifying only about 20,000 protein-coding genes. This is much fewer than the estimated number of proteins in a cell, which raises questions about how so few genes can code for hundreds of thousands of different proteins in a cell and to what extent junk DNA contributes to their regulation, such as switching them on and off.

In the last decade, new methods to identify the DNA that is transcribed into RNA (a chemical cousin of DNA) have suggested that about 80% of DNA may serve some purpose. Many thousands of new hypothetical genes that encode only RNA, but not proteins, have been discovered. Some of these strands of RNA are indeed involved in the regulation of genes, such as deciding when to switch them on and when to switch them off.

The result of these findings made some question the very existence of junk DNA. Yet others argued that the variable size of many genomesfilled with largely repetitive sequences can’t explain the complexity of organisms and that there has to be a chunk of DNA with no function. Why does an onion need a genome that is about five times larger than ours?

The dynamic genome

Although we are now certain that many non-coding DNA sequences are pivotal in protecting and stabilising the genome, regulating genes, differentiating cells and forming tissue, organ development from birth to death, differences between people, their variable response to drugs and other environmental cues, and predisposition to a growing number of human diseases, we do not know how much junk is in our DNA. But can we find out?

To appreciate the origin and extent of junk DNA in our cells, we need to understand how it evolved. One of the most critical events in evolution – the duplication of genes, or their coding parts (called exons) – give the cells and organisms a chance to test new function without endangering their viability or fitness. As duplicated genes, exons or non-coding DNA diverge through errors in replication or DNA repair over many years, the functions of either new or ancestral copies may change. The cell may select sequences underlying new, similar or even opposite functions, leaving either copy in the genomic scrapyard. This does not mean, however, that the discarded DNA segments are no longer useful to the cell.

Genomes are extremely dynamic entities: new functional elements continuously appear and old ones may become extinct. This can be illustrated by repetitive elements named “Alus” that are found in primate genomes, that have accumulated a total of over one million copies and occupy about 11% of human DNA. Alus are often transcribed as RNA and are an important source of new coding parts, gene regulatory elements and protein diversity, especially in highly organised tissues such as the brain. They had a key role in human development and most likely contribute to our distinction from other primates. They might also help evolution get better over time, yet these and other interspersed repeats were initially dismissed as junk.

Are we ready to edit the human genome?

If junk DNA could alter the function of a cell at any time and we can’t define it, how can we safely edit our genomes? Genome editing technologies such as CRISPR are powerful tools to manipulate both coding and non-coding DNA and study their function. But we cannot exclude that unintended changes to what simply looks like junk DNA would not harm how genes are expressed. They may have inadvertent consequences for the cell or the organism that may become apparent only later in life, such as infertility, mental illness or cancer, and propagate into future generations.

For example, manipulating gene-intervening sequences can modify interactions between proteins. Although current genome editing procedures, including gene editing in human embryos, aim at surgically accurate changes of DNA they can’t yet fully exclude undesirable alterations in our non-coding or junk DNA reservoir.

It’s deeply rooted in our nature to fear or dismiss what we don’t fully understand. Although we can’t predict which DNA segment may become functional today and which tomorrow, we are now equipped with formidable tools including genome editing to examine the function of non-coding DNA in greater detail in the coming years. We should support activities that improve this understanding and avoid those that may damage what we may never be able to repair or create again, namely, the irreplaceable heritage of well over one billion years of evolution, including the human genome.


This article was originally published on Nature.  Read the original article.

Death Be Not Programmed

Necrosis is not subtle. One of the body’s natural forms of cell death, it can be likened to a ship hitting an iceberg, exploding the boiler, and capsizing, all at once. Cell necrosis occurs as a result of sudden death by trauma, by pathogenic infection, or by the proverbial last straw laid on accumulated damage.

Underneath all that seeming chaos, however, cell necrosis is actually an ordered chain of steps, says Jeffery Molkentin, an HHMI investigator at Cincinnati Children’s Hospital Medical Center. Moreover, Molkentin and others are crafting treatments for several “untreatable” degenerative diseases by uncoupling key steps that drive necrosis forward.

Blocking a Protein’s Punch

Cyclophilin D isn’t just important in Duchenne MD, but a variety of other diseases.

Although he made his first discovery in the field of necrosis while studying damaged heart cells, Molkentin is focusing on Duchenne muscular dystrophy (MD). The most common form of the genetic muscle-wasting disease affects one in every 3,500 males born in the United States; boys with Duchenne MD typically live only into their late teens and early 20s.

Molkentin has used engineered transgenic mice to show how the relentless muscle damage that drives Duchenne MD can be interrupted. Now, he has a “proof of principle” drug study underway in Brazil, using a small colony of golden retriever dogs that spontaneously develop an aggressive form of MD similar to human Duchenne MD. The drug is related to the immune suppressant cyclosporine, but instead of targeting the immune system it blocks a crucial step in the cell necrosis pathway in skeletal muscles, according to Molkentin.

This drug does not correct the genetic defect that drives MD—mistakes in a giant muscle-connecting protein called dystrophin—so it is no cure for the disease. But stopping cell necrosis could curtail MD’s crippling effects. Though results are not in, Molkentin believes that “if this drug fixes the golden retrievers, it’s going to work in kids.”

The Mitochondrial Link

Molkentin came to the idea of blocking cell necrosis through his earlier studies of cardiac hypertrophy, the enlargement of heart muscle cells, or cardiomyocytes, that follows an ischemic event—a short cut-off of blood to the heart. Cardiomyocytes can’t renew themselves, so cellular necrosis after ischemia leads to heart dysfunction and failure. The tipping point in the cardiomyocyte is the sudden swelling of mitochondria, the cell’s internal power plants. Excess calcium floods in through a regulated pore, finally bursting the organelles, blacking out energy production and spewing toxic proteins.

Molkentin discovered that he could stop mitochondrial swelling by blocking the action of cyclophilin D, a protein that controls the so-called mitochondrial permeability transition pore, or MPTP. Without cyclophilin D, the MPTP stayed firmly shut.

It was startling, says Molkentin, that mouse cardiomyocytes with their cyclophilin D neutralized were protected from mitochondrial swelling and death due to calcium overload. There was something here beyond a vulnerable protein, he realized. Cyclophilin D appears to be one player in a network of proteins that create an ordered process that he calls programmed cell necrosis.

These mice that lack the gene producing laminin 2 have full-blown MD, are dramatically undersized, barely move, and are close to death.

Jeffery Molkentin

To explore programmed necrosis in a living animal, Molkentin created a mouse model lacking the gene Ppif, which produces cyclophilin D. The mouse sailed through laboratory-induced ischemic events, prompting Molkentin to wonder if cell necrosis could be blocked in a genetic disease like MD, where damage builds up over time.

The gene for human Duchenne MD, identified in 1986, produces a defective version of dystrophin, which, along with its helper proteins, normally passes through the sarcolemma, the membrane sheath around skeletal muscle fibers.

In Duchenne MD, defective dystrophin causes the sarcolemma to tear. Calcium pours through the torn membrane and into the skeletal muscle cells. Unable to tolerate the extra calcium, the cells’ mitochondria swell and burst. Cell necrosis then spreads from cell to cell through the long bundles of skeletal muscle fibers.

Because of this mitochondrial link, Molkentin decided to crossbreed his cyclophilin D-null mouse with mice carrying engineered genetic defects that model MD.

In his office at Cincinnati Children’s, Molkentin pulls up a video on his computer. “Look here,” he says. “These mice that lack the gene producing laminin 2 have full-blown MD, are dramatically undersized, barely move, and are close to death.” Molkentin’s double-null mouse—null for laminin 2 and cyclophilin D—has an awkward gait, but it’s mobile and lives well into adulthood. The double-null mouse is living with MD because cell necrosis is stalled.

But mice are mice, Molkentin concedes. If the drug being tested in Brazil over the next year can do this for the golden retrievers, he says, there will be good reason to move toward clinical trials with children.



This article was originally published on HHMI Bulletin.  Read the original article.

CRISPR-like ‘immune’ system discovered in giant virus

Gigantic mimiviruses fend off invaders using defences similar to the CRISPR system deployed by bacteria and other microorganisms, French researchers report1. They say that the discovery of a working immune system in a mimivirus bolsters their claim that the giant virus represents a new branch in the tree of life.

Mimiviruses are so large that they are visible under a light microscope. Around half a micrometre across, and first found infecting amoebae living in a water tower, they boast genomes that are larger than those of some bacteria. They are distantly related to viruses that include smallpox, but unlike most viruses, they have genes to make amino acids, DNA letters and complex proteins.

That means that they blur the line between non-living viruses and living microbes, says Didier Raoult, a microbiologist at Aix-Marseille University in France, who co-led the study with microbiologist colleague Bernard La Scola. Raoult says that he doesn’t consider the mimivirus to be a typical virus; instead, it is more like a prokaryote — microbes, including bacteria, that lack nuclei.


Like prokaryotes, mimiviruses are plagued by viruses known as virophages, Raoult, La Scola and their colleagues reported in 20082. Six years later, in 2014, they found a virophage — named Zamilon — that infects some kinds of mimivirus but not others3. Raoult hypothesized that these infections, which sap a mimivirus’s capacity to copy itself, could have led to the evolution of a defence system much like CRISPR.

Immune defence

In bacteria and another kind of prokaryote, called archaea, CRISPR systems store a library of short DNA sequences that match those of phages and other invading DNA. When a foreign DNA sequence with matching sequences in this library attacks a cell, specialized ‘Cas’ enzymes unwind the intruder DNA and chop it into pieces, stopping an infection. Biologists have now repurposed CRISPR as a technology to edit genomes.

To determine whether mimiviruses have a similar defence system, Raoult’s team analysed the genomes of 60 mimivirus strains and looked for sequences that match those of the Zamilon virophage. Mimiviruses that were resistant to Zamilon also harboured a short stretch of DNA that matched that of the phage.

Adjacent to these sequences, Raoult’s team found genes encoding enzymes that can degrade and unwind DNA. In CRISPR immunity, too, the genes encoding the Cas enzymes sit beside the sequences that recognize the virus. Blocking activity of different components of the system made the mimiviruses susceptible to Zamilon virophage attack. The findings were published on 29 February in Nature1.

It makes sense for mimiviruses to have an immune system because they must compete for resources against against other microbes and viruses, says Raoult. “They are facing the same kind of challenge that prokaryotes have when they live in communities: they need to fight against viruses and prokaryotes. I even suspect they secrete antibiotic compounds.”

Raoult has argued, somewhat controversially, that mimiviruses constitute a fourth domain of life — alongside bacteria, archaea and eukaryotes. He sees their defence system, which he has named MIMIVIRE, as a very ancient adaptation that further supports them having their own branch on the tree of life.

Francisco Mójica, a microbiologist at the University of Alicante in Spain, who identified CRISPR sequences in prokaryotes in the 1990s, notes that CRISPR components have been found in other viruses, but it is not clear whether the systems function. He suspects that an ancestor of mimiviruses picked up MIMIVIRE from another microbe. “It will certainly be of great interest to identify the mechanism involved in MIMIVIRE immunity,” says Mójica; he expects that it will be very different from CRISPR.

Luciano Marraffini, a bacteriologist at the Rockefeller University in New York, says that Raoult’s team makes a good case that MIMIVIRE is a viral defence system, but agrees that it will be important to work out how it stops virophage infections.

Just as unravelling how CRISPR immunity works led to its repurposing as a genome-editing tool, studying mimiviruses could hold surprises, Marraffini says. “The giant viruses most likely enclose a whole lot of new biology, some of which, including the MIMIVIRE, could find novel application. Maybe in genome editing, maybe in other fields.”



This article was originally published on Nature.  Read the original article.

‘Selfish’ DNA flouts rules of inheritance

In the Star Wars movies, the droid R2D2 is a heroic rebel. In living animals, a selfish bit of DNA called R2d2is an outright lawbreaker. It violates laws of both genetic inheritance and Darwinian evolution. R2d2 can sweep through mouse populations by mimicking helpful mutations while actually damaging fertility, researchers report online February 15 in Molecular Biology and Evolution.

The new findings suggest that even genes that hurt an organism’s evolutionary chances can cheat their way to the top. That could be good news for researchers hoping to use engineered “gene drives” to eliminate mosquito-borne diseases and invasive species. But it’s also a cautionary tale for scientists looking for signs that natural selection has picked certain genes because they offer an evolutionary benefit.

If researchers aren’t careful, they may be hoodwinked into thinking that a selfish gene is one that has some evolutionary advantage, says Daven Presgraves, an evolutionary geneticist at the University of Rochester in New York. The genetic signatures are the same, he says. But what looks like survival of the fittest may actually be a cheater prospering.

Geneticist John Didion and colleagues examined DNA samples from wild mice from Europe and North America to determine how widespread R2d2 has become. The researchers also bred strains of mice in the lab to determine how quickly R2d2 is capable of spreading. The selfish DNA could blaze through populations, reports Didion, formerly of the University of North Carolina at Chapel Hill. The proportion of mice with the selfish gene more than tripled in one laboratory population from 18 percent to 62 percent within 13 generations, the researchers found.

In another breeding population, R2d2 shot from being in 50 percent of the lab mice to 85 percent in 10 generations. By 15 generations, the selfish element reached “fixation” — all the mice in the population carried it. That rate of spread was much faster than Didion, now at the National Human Genome Research Institute in Bethesda, Md., and colleagues predicted. Computer simulations had projected it would take 184 generations for the selfish DNA to spread to all of the mice. 

Such wildfire spread of a gene variant that eventually wipes out all other versions is known as a selective sweep. Sweeps are hallmarks of a gene that helps an organism adapt to its environment. But this study suggests that what looks like adaptation may actually be selfish genetics at work, says Nitin Phadnis, an evolutionary geneticist at the University of Utah in Salt Lake City.

R2d2 is a “selfish element,” a gene or other piece of DNA that causes itself to be inherited preferentially, researchers at UNC Chapel Hill reported last year. The droid’s namesake is a stretch of DNA on mouse chromosome 2 that contains multiple copies of the Cwc22 gene. When seven or more copies of that gene build up on the chromosome, R2d2 gets selfish. In female mice, it elbows aside the chromosome that doesn’t contain the selfish version of the gene and is preferentially incorporated into eggs. That’s a violation of the laws of inheritance spelled out by Gregor Mendel in which each gene or chromosome is supposed to have a fifty-fifty chance of being passed on to the next generation. But there is a cost toR2d2’s selfishness: Female mice that carry one copy of the selfish element have small litter sizes compared with mice that don’t carry the greedy DNA.

Under evolutionary laws, that loss of fertility should cause natural selection to weed out R2d2. But the selfish element’s greed is greater than the power of natural selection to combat it, the lab experiments show.

Even the most successful cheat can get caught, though. Wild mice in Europe and at two sites in the United States had widely varying proportions of the selfish gene, Didion and colleagues found. In a population of German mice, R2d2 was in 67 percent. But only 8 percent of Greek mice had the selfish DNA. In Maryland, where mice carrying R2d2 were first found, 21 percent of mice carry it. The selfish element hasn’t crossed the country yet. None of the mice the researchers tested from California had R2d2.

Based on lab experiments, the researchers might have expected all the wild mice they tested to carry the deceptive DNA. The relatively low proportion of wild mice carrying R2d2 could mean that some mice have developed ways to suppress the gene’s selfishness, says Matthew Dean, an evolutionary geneticist at the University of Southern California in Los Angeles.



This article was originally published on Science.  Read the original article.

DNA Under the Scope, and a Forensic Tool Under a Cloud

Marina Stajic worked for nearly three decades as director of the forensic toxicology lab at the medical examiner’s office in New York City. Last week Dr.. Stajic, 66, filed a lawsuit against the city, claiming she had been forced into retirement last year in part because of a disagreement with her superiors over the accuracy of certain DNA tests.

There is more at stake here than Dr. Stajic’s retirement. The cutting-edge technique at the center of this legal dispute, called low copy number DNA analysis, has transformed not just police work, but also a range of scientific fields including cancer biology, in vitro fertilization, archaeology and evolutionary biology. Yet some of the technique’s applications have triggered scientific controversy.

The medical examiner’s office has become a strong advocate for the technique. It is the only public lab in the United States that uses low copy number DNA to develop profiles for use in criminal cases. But experts have long warned that investigators must take particular care in interpreting these tests: analyzing so few DNA molecules can lead to errors.

When scientists first began deciphering DNA in the 1970s, they needed large amounts, because the chemical processes that were involved destroyed most of the source material.

In 1983, the NobelPrize-winning biochemistKary Mullis sped up the process with a kind of photocopying machine for DNA called polymerase chain reaction, or PCR. Dr. Mullis showed the world how to make millions of copies of any particular genetic fragment.

PCR made it possible for scientists to work with DNA in smaller samples, since they could now make more of it. Over the past four decades, researchers have come up with ways to run ever-more-sensitive tests. In the most extreme of these, experts can reconstruct the entire genome using DNA fragments extracted from a single cell.

Similar tests allow scientists to study DNA from fossils dating back thousands of years. Over time, genetic material in fossils degrades until it becomes scarce. Yet scientists only need to rescue trace amounts for analysis.

“We can be down to 10 or 20 molecules,” said Brian Kemp, a geneticist at Washington State University, who regularly extracts DNA from tiny fish vertebrae more than 10,000 years old.

Scientists have found a number of ways to make their tests more sensitive. For instance, Dr. Kemp and his colleagues have discovered that collagen and other compounds found in fossils can slow down the PCR process. By carefully removing those chemicals, they are able to make more copies of ancient DNA.

But the more sensitive DNA tests become, the greater the risk that they will yield the wrong result. Even stray bits of DNA — from a lab worker’s skin cell or an airborne fungal spore — may contaminate the test equipment.

When scientists then run PCR reactions, they may make millions of copies of the contaminating DNA along with the genetic material they want to study. Even a little contamination can skew results.

Scientists have developed many safeguards to prevent contamination. Stephen R. Quake, a biologist at Stanford University, and his colleagues have shrunk down the equipment they use, so that there is less room for contaminating cells to invade.

“Whatever’s floating around in a test tube, you’re going to have a thousand times less of it,” Dr. Quake said, referring to the miniaturized setup.

Like other scientists, forensic researchers also are using less and less DNA. Now investigators can get usable genetic material just by wiping gun grips or other surfaces for a few loose skin cells.

But low copy DNA analysis can detect a mix of DNA from more than one person, and it can be hard to tell which of them is relevant to a crime.

“Maybe there’s not three people bleeding on a steering wheel, but there are three people touching it,” said Kirk E. Lohmueller, a geneticist at the University of California, Los Angeles. “Before, you didn’t have to worry about that.”

People can even leave DNA traces on objects they haven’t touched. In the January issue of The International Journal of Legal Medicine, German researchers vividly illustrated this problem.

They rubbed a cloth on people’s necks, and then gave the cloths to a second group of people. The second group rubbed their hands on the cloth, picking up the DNA from the first group, then handled a plastic bag or a cotton cloth.

When the scientists examined the bags and cloths, they found DNA from the first group of subjects about 40 percent of the time.

Low copy number DNA analysis can also give incorrect results when it pushes PCR chemistry beyond its limits. There can be so few DNA molecules floating around in a test tube that they simply don’t bump into the PCR chemicals needed to replicate them. As a result, some of the original DNA may go unduplicated.

This failure can yield a genetic profile that doesn’t match the source of the DNA.

Each of us carries two copies of every gene. Sometimes the copies are identical, but not always. If a suspect has two different versions of a gene and PCR duplicates just one, then a forensic scientist may conclude both copies are identical.

This error, called allelic drop-out, doesn’t mean that low copy number DNA is useless. Statisticians are developing methods to calculate the reliability of PCR results for each gene. Those methods should help investigators figure out how confident to be in their overall analysis.

Last July, defense lawyers in a Brooklyn murder trial challenged DNA evidence that came from a bicycle handle. They offered testimony from experts who criticized the medical examiner’s methods, such as the way they calculated the odds of allelic drop-out. The judge threw out the DNA results.

Bruce Budowle, the executive director of the Institute of Applied Genetics at the University of North Texas Health Science Center, served as an expert witness for the defense. (His fee was put toward his university’s program for student stipends.) Despite delivering harsh criticisms of the medical examiner’s office on the stand, he sees a lot of promise in low copy number DNA analysis.

He’s using it in his own research, which includes identifying remains of Civil War soldiers and skeletons discovered in Deadwood, S.D. And Dr. Budowle, like other experts, is working on new methods to improve the technique’s sensitivity.

The problem, he said, is that criminal investigations leave far less room for error.

“I’m not pleased with what’s been done with low copy number in forensics to date,” he said. “But if we get better interpretation, I think it could be a better system.”



This article was originally published on The New York Times.  Read the original article.