Faster, Cheaper DNA Sequencing Method Devised

ScienceDaily (Dec. 22, 2009) — Boston University biomedical engineers have devised a method for making future genome sequencing faster and cheaper by dramatically reducing the amount of DNA required, thus eliminating the expensive, time-consuming and error-prone step of DNA amplification.

A team of researchers led by Boston University biomedical engineer Amit Meller is using electrical fields to efficiently draw long strands of DNA through nanopore sensors, drastically reducing the number of DNA copies required for a high throughput analysis. (Credit: Figure copyright, Nature Nanotechnology, 2009)

In a study published in the Dec. 20 online edition of Nature Nanotechnology, a team led by Boston University Biomedical Engineering Associate Professor Amit Meller details pioneering work in detecting DNA molecules as they pass through silicon nanopores. The technique uses electrical fields to feed long strands of DNA through four-nanometer-wide pores, much like threading a needle. The method uses sensitive electrical current measurements to detect single DNA molecules as they pass through the nanopores.

“The current study shows that we can detect a much smaller amount of DNA sample than previously reported,” said Meller. “When people start to implement genome sequencing or genome profiling using nanopores, they could use our nanopore capture approach to greatly reduce the number of copies used in those measurements.”

Currently, genome sequencing utilizes DNA amplification to make billions of molecular copies in order to produce a sample large enough to be analyzed. In addition to the time and cost DNA amplification entails, some of the molecules — like photocopies of photocopies — come out less than perfect. Meller and his colleagues at BU, New York University and Bar-Ilan University in Israel have harnessed electrical fields surrounding the mouths of the nanopores to attract long, negatively charged strands of DNA and slide them through the nanopore where the DNA sequence can be detected. Since the DNA is drawn to the nanopores from a distance, far fewer copies of the molecule are needed.

Before creating this new method, the team had to develop an understanding of electro-physics at the nanoscale, where the rules that govern the larger world don’t necessarily apply. They made a counterintuitive discovery: the longer the DNA strand, the more quickly it found the pore opening.

“That’s really surprising,” Meller said. “You’d expect that if you have a longer ’spaghetti,’ then finding the end would be much harder. At the same time this discovery means that the nanopore system is optimized for the detection of long DNA strands — tens of thousands basepairs, or even more. This could dramatically speed future genomic sequencing by allowing analysis of a long DNA strand in one swipe, rather than having to assemble results from many short snippets.

“DNA amplification technologies limit DNA molecule length to under a thousand basepairs,” Meller added. “Because our method avoids amplification, it not only reduces the cost, time and error rate of DNA replication techniques, but also enables the analysis of very long strands of DNA, much longer than current limitations.”

With this knowledge in hand, Meller and his team set out to optimize the effect. They used salt gradients to alter the electrical field around the pores, which increased the rate at which DNA molecules were captured and shortened the lag time between molecules, thus reducing the quantity of DNA needed for accurate measurements. Rather than floating around until they happened upon a nanopore, DNA strands were funneled into the openings.

By boosting capture rates by a few orders of magnitude, and reducing the volume of the sample chamber the researchers reduced the number of DNA molecules required by a factor of 10,000 — from about 1 billion sample molecules to 100,000.

The research was funded by the National Human Genome Research Institute of the Institutes of Health and by the National Science Foundation.

DNAWellnessinfo.com Resource:  http://www.sciencedaily.com/releases/2009/12/091220143923.htm

Scientists use DNA sequencing to attack lung cancer

DALLAS — Dec. 16, 2009 — Aided by next-generation DNA sequencing technology, an international team of researchers has gained insights into how more than 60 carcinogens associated with cigarette smoke bind to and chemically modify human DNA, ultimately leading to cancer-causing genetic mutations.

In a new study available online and in a future issue of the journal Nature, lung-cancer experts in the Harold C. Simmons Comprehensive Cancer Center at UT Southwestern Medical Center worked with scientists from the Cancer Genome Project in the United Kingdom to determine the entire genetic sequence of cancer cells from a patient with small-cell lung cancer (SCLC). They then compared those results with normal DNA isolated from the same patient.

Using new DNA sequencing technology called “massively parallel sequencing,” the researchers searched the DNA sequences for differences between tumor and normal cells. They found more than 23,000 mutations that the tumor cells had acquired and also discovered a new gene involved in lung cancer named CHD7.

The number of mutations from the study suggests that a person may develop one mutation for every 15 cigarettes smoked, said Dr. John Minna, director of the Nancy B. and Jake L. Hamon Center for Therapeutic Oncology Research at UT Southwestern and one of the authors of the new study.

The researchers said the findings illustrate the power of advanced technology to provide important new information about human cancer, including the effect of cancer-causing chemicals on the body and the identification of potential new therapeutic targets.

“Cancer is driven by acquired mutations in genes, and we are at a point where it soon will be possible to actually know every mutation in the tumors of each of our patients,” Dr. Minna said.

“The key will be to use this information to find new ways to help prevent cancers, diagnose them earlier and to select treatments that might be specific for each patient’s tumor. While these findings are the first step, they have lighted our path to clearly point us in the right direction. In addition, they provide the first detailed analysis of a human cancer — lung cancer ? that is closely linked to smoking.”

Dr. Minna and Dr. Adi Gazdar, professor of pathology in the Hamon Center at UT Southwestern, provided the SCLC cells and normal cells for the research. Dr. Minna, who also directs the W.A. “Tex” and Deborah Moncrief Jr. Center for Cancer Genetics, and Dr. Gazdar have developed one of the most extensive collections of lung-cancer cell lines, which are used by researchers worldwide in studies of the disease. The SCLC and normal cells used in the study are designated NCI-H209 and NCI-BL209, respectively, and were established from a patient Drs. Minna and Gazdar treated 30 years ago.

When the researchers analyzed the 23,000 mutations, they found distinctive patterns associated with the cocktail of carcinogens present in cigarette smoke. The DNA sequence of the cancer cells also revealed that the cells had attempted to repair their smoke-damaged DNA using two mechanisms, but the cells were only partially successful.

Cigarette smoke deposits hundreds of chemicals into the airways and lungs. The longer one smokes and the more cigarettes smoked each day, the higher the risk of developing lung cancer and mutations.

“By applying the same approach to other cancers not associated with cigarette smoking, including the very large group of people who develop lung cancer but have never smoked, it may be possible to discern which carcinogens play a role in those other cancers as well,” Dr. Gazdar said.

Dr. Minna added that the research methods used to analyze the cancer cells represented a technological tour de force.

“The data demonstrate the power of whole-genome sequencing to untangle the complex mutational signatures found in cancers induced by cigarette smoke,” Dr. Minna said. “In addition, the protein product of the CHD7 gene now becomes a new marker for early diagnosis and also for potentially targeted therapy.”

Lung cancer is the leading cause of cancer-related deaths worldwide, developing in more than a million patients annually. People who smoke are 10 to 20 times more likely to get lung cancer or die from lung cancer than people who do not smoke. SCLC represents 15 percent of these cases and is associated with early metastasis, relapse after initial response to chemotherapy and less than a two-year survival rate.

The study was supported by grants from the Wellcome Trust, the Human Frontiers Science Program and the National Cancer Institute.

Visit www.utsouthwestern.org/cancercenter to learn more about UT Southwestern’s clinical services in cancer.

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Experts To Discuss DNA Barcodes And Their Uses

Article Date: 09 Nov 2009 – 2:00 PST – medical news today

World experts are gathering this week to discuss DNA barcodes and their uses, covering a wide range of areas from medicine to agriculture, health to fraud, from smuggling to exploring our planet’s prehistoric life.

About 350 experts from 50 countries are meeting for the third International Barcode of Life conference that is taking place from 9 to 11 November in Mexico City.

DNA barcoding is a new technique that uses a short DNA sequence from the genome of an organism, living or dead, as a molecular way of identifying the species it belongs to. DNA barcode sequences are very short compared to the entire genome and can be obtained quite quickly and cheaply.

The Consortium for the Barcode of Life (CBOL) is an international initiative devoted to developing DNA barcoding as a global standard for the identification of biological species.

Through the CBOL initiative, experts are agreeing a standard for DNA barcoding.

The challenge for the initiative is finding an area of DNA that does not vary much down generations, yet varies sufficiently between species to make identification reliable. A number of studies have shown that for higher animals, the variability of the “Folmer region” at the 5′ end of the cytochrome c oxidase subunit 1 mitochondrial region (COI) is very low (about 1 to 2 per cent) and even between closely related species it differs by several per cent, making this the ideal region on which to settle as the standard for DNA barcoding.

This section of DNA is 648 nucleotide pairs long for most groups and is surrounded by regions that are reasonably conserved, making it quite easy to isolate and analyze.

In some groups, COI is not an effective barcode region and a different standard region will have to be sought and agreed on. But the idea is that in all cases, DNA barcoding uses a short, standard region that enables cost-effective species identification.

As the standard is being thrashed out and discussed, all manner of professionals are starting to get interested in its application, from medical and agricultural researchers, to police and customs officers.

For instance, using DNA barcoding, palaeontologists hope to be able to sequence ancient plant and animal remains extracted from degraded DNA in northern permafrost cores to reveal Earth’s pre-historic life, and how life on Earth responded to global climate change.

And by analyzing the DNA of gut contents, scientists hope to discover secrets of what eats what in the animal world.

One such group is the The International Barcode of Life Project, headquartered in Guelph, Canada, where barcoding was pioneered. They will be telling meeting delegates about their discovery that eight species of bat feed on over 300 types of insect, one of the largest food webs ever found.

Conservationists are now getting very excited about the application of DNA barcoding to help unravel the complexity of the dynamics in the natural world.

Scott Miller, Acting Under Secretary for Science at the Smithsonian Institution and Chair of the Consortium for the Barcode of Life (CBOL), who are co-hosting the meeting with the Instituto Biologia, Universidad Nacional Autonoma de Mexico (UNAM), told the press that:

“DNA barcoding is opening a new window into the relations between hunter and prey in the wild and how diets may be changing due to climate change.”

He explained that like gut contents, soils contain a mixture of species that are hard to identify using convetional tools. Tiny soil organisms eat each other, they eat roots, and all sorts of animal and plant debris, so:

“Knowing what eats what is important to many studies, including investigations into how much carbon dioxide and other greenhouse gases are being released from soils into the atmosphere,” said Miller.

Another area of application would be producing evidence to prosecute smugglers of wild bushmeat and other products made from endangered species: a trade that last year netted 15 billion dollars worldwide.

When smoked or sundried, only DNA barcoding can differentiate bushmeat from domestic animal meat like beef, goat or pork, so law enforcement agencies are becoming increasingly interested in the DNA barcode library of endangered species that Dr George Amato of the American Museum of Natural History in New York is compiling.

The hope is that the Mexico meeting will bring about a global agreement on how to do the same with plants, which would for instance help to track down illegal timber trading and regulate herbal medicines, among others.

CBOL Executive Secretary David Schindel said:

“Biodiversity scientists are using DNA technology to unravel mysteries, much like detectives use it to solve crimes. It is having a profound impact on our understanding of organisms in nature and how they interact with the environment.”

Following increases in the number of puffer fish poisoning cases in the US due to fradulent food labelling, the US Food and Drug Administration (FDA) will be telling delegates about their interest in DNA barcoding and the challenge posed by trying to differentiate among different species in marketed seafood, an increasing proportion of which is now imported, and which is also processed to “a point where traditional morphologic species determination is not possible”.

An FDA representative told the press that:

“New methods that allow accurate and rapid species identifications are critical for both food borne illness investigations and for the prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value.”

The FDA will also be presenting a study that showed DNA barcoding reliably distinguished the seedpods of Star Anise ( Illicium verum, a herb used in teas, herbal remedies and cooking) from otherwise identical seedpods of a sister species, Illicium anisatin, considered to contain neurotoxic compounds and therefore a health risk.

The delegates will also hear of a case from Canada, where students nationwide collected fish samples from stores and analyzed the resulting DNA data, revealing significant market “mislabelling” of seafood.

Another case that will be presented will be the successful apprehension of a Brazilian smuggler last year who was caught trying to smuggle parrot eggs which he said were quails’ eggs, but DNA barcoding revealed that they were the eggs of several species of parrots and macaws, many of which where either threatened or vulnerable.

A medical application of DNA barcoding will help to identify black flies in Brazil and other South American countries where they spread river blindness disease. So far 70 species of black flies have been barcoded to date, about 20 per cent of the number known to science, and including three previously unrecognized.

Other medical applications include identification of malaria mosquitoes in India, parasite bearing freshwater snails in the Cameroons, nematode parasites in Mexico that attack crops, humans and livestock.

Mexico is one of the countries that is moving ahead quickly in using DNA barcoding. Under the auspices of CONACYT, Mexico’s National Council on Science and Technology, they have established a national barcode network (MexBOL) involving 60 researchers from 15 institutions.

Mexico now has a number of new “barcode factories” at institutions in the north, center and south of the country, including CIBNOR (Centro de Investigaciones Biológicas del Noroeste), IBUNAM (Instituto de Biología, UNAM), and ECOSUR (El Colegio de la Frontera Sur).

Meeting co-host Patricia Escalante, chair of the Zoology Department, Institute of Biology, UNAM, said this work in Mexico and elsewhere was very important.

“Barcoding is a tool to identify species faster, more cheaply, and more precisely than traditional methods,” she explained.

MexBOL will produce barcodes for all important taxonomic groups including national campaigns, such as barcoding all trees (ArBOL), fungi, bees, aquatic insects, crayfishes, fishes, birds, mammals and more.

Escalante explained that:

“We need an accurate inventory of global biodiversity to recognize parasites of medical, economic or ecological importance.”

“This work will help develop biological control measures, monitor and control of human diseases and potential zoonoses, manage agricultural and aquaculture pathogens, and detect the presence of invasive species,” she added.

The largest barcode factory in the world is at the Biodiversity Institute of Ontario at the University of Guelph in Canada, where DNA barcoding was first proposed and developed.

Similar facilities are being set up at the French Museum National d’Histoire Naturelle, as well as in the Netherlands and Poland.

– 3rd International Barcode of Life Conference

Source: Consortium for the Barcode of Life (CBOL).

Written by: Catharine Paddock, PhD
Copyright: Medical News Today

DNAWellnessinfo.com Resource:  http://www.medicalnewstoday.com/articles/170276.php