GeneWatch Vol. 26 No. 5

GeneWatchTHE MAGAZINE OF THE COUNCIL FOR RESPONSIBLE GENETICS | ADVANCING THE PUBLIC INTEREST IN BIOTECHNOLOGY SINCE 1983

Volume 26 Number 5 | NoV-Dec 2013

ISSN 0740-9737

Rob DeSalle Science, Plain and BeautifulDavid Schindel on the Barcode of Wildlife project

Jonathan Deeds on identifying mislabeled seafood

Featuring:

November-December 2013 2 GeNeWatch

GeneWatch is published by the Council for Responsible Genetics (CRG), a national, nonprofit, tax-exempt organization. Founded in 1983, CRG’s mission

is to foster public debate on the social, ethical, and environmental implications of new genetic technologies. The views expressed herein do not necessarily represent

the views of the staff or the CRG Board of Directors.

aDDress 5 Upland Road, Suite 3 Cambridge, MA 02140 PhoNe 617.868.0870 Fax 617.491.5344

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boarD of Directors

shelDoN KrimsKy, PhD, boarD chair Tufts University

eVaN balabaN, PhDMcGill University

Paul billiNgs, mD, PhDLife Technologies Corporation

robert Desalle, PhD

American Museum of Natural History

robert greeN, mD, mPhHarvard University

Jeremy gruber, JDCouncil for Responsible Genetics

rayNa raPP, PhDNew York University

Patricia Williams, JDColumbia University

staff

Jeremy Gruber, President and Executive DirectorSheila Sinclair, Manager of Operations

Samuel Anderson, Editor of GeneWatchAndrew Thibedeau, Senior Fellow

Vani Kilakkathi, Fellow

coVer DesigN Samuel W. Anderson

eDitorial & creatiVe coNsultaNt

Grace Twesigye

GeneWatchNovember-December 2013

volume 26 Number 5

eDitor aND DesigNer: Samuel W. AndersoneDitorial committee: Jeremy Gruber, Sheldon Krimsky,

Ruth Hubbard

Unless otherwise noted, all material in this publication is protected by copyright by the Council for Responsible Genetics. All rights reserved. GeneWatch 26,5

0740-973

The Council for Responsible Genetics is saddened by the pass-ing of Adrienne Asch on November 19. A former member of the CRG Board of Directors, Adrienne was most recently director of the Center for Ethics at Yeshiva University and the Edward and Robin Milstein Professor of Bioethics as well as professor of epi-demiology and population health and family and social medicine at Albert Einstein College of Medicine.

A pioneer in disability studies, Adrienne was always trying to change the all too frequent belief that disability was tragedy rath-er than just another aspect of human life and maintained that the rights of disabled women should be as much a feminist concern as those of able-bodied ones.

Her work was devoted to the ethical, political, psychological, and social implications of human reproduction and the family. She produced tremendous scholarship that stood at the nexus of bioethics, disability studies, reproductive rights and feminist theory. Her publications include two volumes of which she was a co-editor: Women with Disabilities: Essays in Psychology, Cul-ture, and Politics (1988, with Michelle Fine) and Prenatal Testing and Disability Rights (2000, with Erik Parens).

Adrienne was fiercely committed to defending the rights of all human beings, particularly the rights of children with disabilities and opposed the use of prenatal testing and abortion to select children free of disabilities. As she wrote in one of her frequent contributions to GeneWatch:

My moral opposition to prenatal testing and selective abor-tion flows from the conviction that life with disability is worth-while and the belief that a just society must appreciate and

In Memoriam: Adrienne Asch

comments and submissions

GeneWatch welcomes article submissions, comments and letters to the editor. Please email [email protected] if you would like to submit a letter or any other comments or queries, including proposals for article submissions.

founding members of the council for responsible geneticsRuth Hubbard • Jonathan King • Sheldon Krimsky

Philip Bereano • Stuart Newman • Claire Nader • Liebe Cavalieri Barbara Rosenberg • Anthony Mazzocchi • Susan Wright

Colin Gracey • Martha Herbert • Terri Goldberg

Continued on page 4

Cover photo: Grace Twesigye

Cover photo by Grace Twesigye

GeNeWatch 3volume 26 Number 5

GeneWatch Vol. 26 No. 54 President’s Note: DNA Barcoding and Misplaced Priorities By Jeremy Gruber

5 DNA Barcoding Ready for Breakout It’s an eminently practical technology that hasn’t received much fanfare – but that may be about to change. By Mark Stoeckle

7 Science, Plain and Beautiful DNA barcoding may not be “sexy,” but it is beautiful in its simplicity and expediency. By Rob DeSalle

9 Citizen Science Easy enough for kids and amateur scientists to use, you might say DNA barcoding is inherently democratic. By Ellen Jorgensen

11 What’s the Holdup? Outside academia – and especially in highly regulated environments – DNA barcoding is only gradually replacing slower, less accurate, and more expensive methods. Which begs the question: Why? By Peter Christey

14 The Barcode of Wildlife Project DNA barcoding takes center stage in the international effort to combat wildlife trafficking. By David Schindel

17 Foiling Poachers With DNA Barcoding In South Africa, where poachers and smugglers continually adapt to stay a step ahead of environmental law enforcement, DNA barcoding is about to be a game changer. By Jacques du Toit

18 Identifying Commercial Seafood DNA barcoding is a valuable tool for FDA for the species identification of seafood. By Jonathan Deeds

19 Uncovering Herbal Fraud With DNA Barcoding How do you know your Gingko biloba is Ginkgo biloba? A new study shows that’s a very valid question (and answers it). By CRG Staff

20 Monitoring Animal Products and Feeds With DNA Barcoding An interview with Haile Yancy, U.S. Food and Drug Administration.

22 Measuring the Health of Aquatic Ecosystems With DNA Barcoding The U.S. Environmental Protection Agency is finding DNA barcoding to be faster, more precise, and more accurate than previous methods of aquatic bioassessment. By Erik Pilgrim

23 Endnotes

Image: S.W

. Anderson


that non-scientists can pursue it for both educational purposes and the common good.

You would think such a fantas-tic tool, developed as a result of the revolution in our understanding of genetics, would receive regular at-tention in both major media and bio-technology-oriented publications.

You’d be wrong.Indeed, in the realm of conserva-

tion genetics, most media and public attention has been drawn in recent months to discussions of resurrect-ing extinct and endangered species through cloning, an eyebrow-rais-ing proposal that has succeeded in grabbing headlines and funding for conferences but which has dubious practical benefits in any near term for conservation. Meanwhile, every day around the world scientists are developing DNA barcoding technol-ogy, building reference libraries, and diversifying its applications.

For these very reasons, this special issue of GeneWatch is devoted to get-ting our priorities straight by offering our readers a comprehensive picture of the benefits of this important tech-nology and the challenges we face in promoting its broader acceptance and use. nnn

Jeremy Gruber, JD, is President of the Council for Responsible Genetics.

As a bioethical organization, the Council for Responsible Genetics of-ten finds itself in the role of the critic. We identify and raise awareness of bad science and the excesses, mis-placed priorities and foolhardiness attendant to reductionist approaches toward developing genetic science and technology. Rarely do we see the need to play the role of cheerleader among a loud and diverse chorus promoting biotechnology develop-ment at all costs.

DNA barcoding is a critical exception.

DNA barcoding is a simple, stan-dardized way of identifying species from a small sample of DNA. In ani-mals, for example, a set of about 650 base pairs is scanned, with unique re-sults for each species. That “barcode” can then be searched against a refer-ence library for a matching barcode, which will tell you what species your sample came from.

DNA barcoding has almost lim-itless potential as both a conserva-tion and consumer protection tool. It can be used to enhance protection of endangered species by aiding in the identification of bushmeat and other animal products. It can be used to conduct detailed biosurveys to identify what lives in a specific area in order to determine whether the ecosystem is in distress, or whether protected or invasive species are present. It can be used to identify falsely labeled consumer products, from sushi to herbal supplements to medicine. And it’s such a simple tool

President’s Note: DNA Barcoding and Misplaced Priorities

By Jeremy GruBer

nurture the lives of all people, whatever the endowments they receive in the natural lottery. I hold these beliefs because there is abundant evidence that peo-ple with disabilities can thrive even in this less than welcoming society. Moreover, people with disabilities do not merely take from others, they contribute as well-to families, to friends, to the economy. They contribute neither in spite of nor because of their disabilities, but because along with their disabilities come other characteristics of personality, talent, and human-ity that render people with dis-abilities full members of the hu-man and moral community.

When recently asked what she saw as the most pressing bio-ethical problem today, Adrienne responded:

There isn’t enough social jus-tice discussion in bioethics, whether it’s about healthcare or about the equality of all people with their different character-istics. I’m looking for a society that respects the uniqueness and the contributions of every individual, and the capacity of each individual to contribute according to their abilities ... and to be provided for accord-ing to their needs. And that’s an old socialist-Marxist notion, but it’s the society I’m interested in creating. And I’d like a bioeth-ics along with a feminism that was interested in creating that. I think that’s out of fashion but that’s really what I’m looking for.

Her sharp mind and her love of ideas will be sorely missed by her friends and colleagues. nnn

Memoriam, continued from p. 2


by analogy to the Uniform Product Code (UPC) for commercial goods, and presciently proposed a universal reference library. Recognizing that the value of DNA barcoding lay in standardization, it was nonetheless evident at the outset that alternative targets or amplification strategies would be needed for certain groups, including plants and fungi, as COI differed little among species in the former and often contained introns (non-coding pieces of DNA) in the latter.

In 2003, the Alfred P. Sloan Foun-dation sponsored workshops at Cold Spring Harbor Laboratory which explored making this vision a real-ity. Participants envisioned a DNA-based standardized identification system of great use to society and science: Organisms in any life stage could be unambiguously named from bits and pieces, including morpho-logic “look-alike” cryptic species, by experts and non-experts, at relatively low cost, and potentially via automat-ed devices. The biggest challenge was how to build the library, which would involve sampling multi-millions of specimens, each of which had to be identified by experts and archived in museums or herbaria so they would be available to be re-examined. New database practices were needed to link sequence records to individual specimens.

The need to organize a wide sci-entific community effort led to the Consortium for the Barcode of Life (CBOL), an international initiative

DNA barcoding is simple enough to be employed by high school stu-dents and versatile enough to help ferret out food fraud, rein in illicit trade in endangered timber, recog-nize disease-carrying mosquitos, re-veal what tiny insects and big mam-mals eat, and speed discovery of new species on land and sea. DNA barcode similarities and differences among species help make plain the story of evolution. In its first decade DNA barcoding racked up these and many other successes. What lies ahead is expansion into routine use by consumers, educators, citizen sci-entists, DIY biologists, and regula-tory agency enforcers.

The roots of DNA barcoding lie deep in evolutionary study and the recognition that the history of life forms is written in their DNA. The manifest story began at the University of Guelph in 2003, when Paul Hebert and colleagues made an imaginative proposal: a universal DNA identifica-tion system for all macroscopic life.1 They focused on a 650 base pair re-gion of mitochondrial cytochrome c oxidase I (COI), previously reported recoverable from diverse animal phyla by polymerase chain reaction using a single primer pair. Accord-ing to the limited data then available, this region varied little within animal species but generally differed among even closely-related species, making it usually straightforward to match sequences to species names.

Hebert and colleagues called this biological identifier a “DNA barcode”

DNA Barcoding Ready for BreakoutIt’s an eminently practical technology that hasn’t received much fanfare – but that may be about to change.By mark Stoeckle


devoted to developing DNA barcod-ing as a global standard for the iden-tification of biological species, inau-gurated in April 2004 with support from the Alfred P. Sloan Foundation. As of 2013, the Consortium has over 200 member organizations from over 50 countries. Participants at CBOL-sponsored workshops agreed on standard loci for animals, plants and fungi.2 The Gordon and Betty Moore Foundation provided crucial early funding that helped establish DNA barcoding as a scientific enterprise.

In parallel to CBOL, major sup-port from Genome Canada and the Ontario Genomics Institute helped launch the International Barcode of Life (iBOL) in 2010 to speed library building and promote regional net-works and taxon and ecosystem campaigns. The University of Guelph built an online database and work-bench, Barcode of Life Datasystems (BOLD). As of October 2013, BOLD contains over 2.5 million barcode re-cords from over 190,000 named ani-mal, plant, and fungal species.

The barcoding initiative has high-lighted how much we don’t know about macroscopic biodiversity. Many barcoded specimens have turned out to represent new species, and many more as yet undescribed species are represented in databases, awaiting formal names. The underly-ing challenge is the immense number of living species: about 2 million de-scribed among an estimated 8 mil-lion existing. For animal COI bar-codes, BOLD recently instituted an automated Barcode Index Number (BIN) system that assigns numbers to clusters of closely related sequences.3 In well-studied groups, BINs usually correspond to species, suggesting this algorithm can also organize re-cords of unidentified specimens into sets representing new taxa and speed formal descriptions.

Although new sequencing tech-nologies may supplant current Sanger standard, a decade of experi-ence says the key elements — namely, agreed-upon standard gene region(s) and high quality sequence records from documented specimens — are irreplaceable.

Much wider use of DNA barcod-ing in everyday applications awaits. Potential arenas include food and herbal product testing, education, and citizen naturalists. Consumers want to know more about what they eat: where it comes from, whether it is healthy for you, and whether it is produced in a way that is good for the environment, for example. At the same time, barcoding uncovers rou-tine mislabeling of diverse foods — in such cases, forget worrying about where it came from, it is not even the species the label says it is! These in-clude fish (1/3 of U.S. fish products are mislabeled), ground meat (horse-meat-in-hamburger scandal in Eu-rope), olive oil, cheese, tea, and pet food, with costs to consumers and threats to the environment.

Investigations to date suggest if a product is expensive and can’t be readily identified by appearance, it is at risk of mislabeling. A recent barcoding study of herbal products, which are morphologically unidenti-fiable even by experts, supports this point — the majority contained con-taminants, substitutions, or fillers.4 Regulatory agencies are adopting this technology, perhaps a prelude to rou-tine use by food and herbal product distributors, enabling certification, supplemented with consumer level testing by agencies or individuals.

In education, DNA barcoding of-fers a relatively simple and widely ap-plicable technology that allows stu-dents to design and carry out diverse investigations. Science is a process of discovery, but most high school

laboratory exercises have pre-deter-mined outcomes. DNA barcoding enables students to make real dis-coveries and contribute to reference databases. Model projects in a few dozen schools, notably in NYC, Cali-fornia, and Ontario, have demon-strated the potential.5-7 These could serve as templates and be expanded to be a routine component of high school biology. Another plus is that comparing DNA barcode sequences to reference databases provides stu-dents a direct look at evolution.

DNA barcoding democratizes ac-cess to knowledge about biodiversity. It can help citizen naturalists and DIY biologists make modern day voyages of bio-discovery in urban, rural, and wild environments. Just as GPS went from a cumbersome research tool to an everyday application, DNA bar-coding has potential appeal to a large, diverse set of interested individuals.

For wider commercial use, such as by food distributors, certified, afford-able, and speedy barcode testing ser-vices are needed. Robust and rapid protocols for DNA extraction, ampli-fication, and sequencing are already available, indicating the hurdles, if any, are in business aspects. For con-sumer, educational, and DIY use, easy-to-use inexpensive kits with mail-in sequencing services would likely boost demand.

We look forward to DNA barcod-ing’s breakout applications. nnn

Mark Stoeckle, MD, is Senior Research Associate in the Program for the Human Environment at The Rockefeller Univer-sity. He has been involved in the DNA Barcoding Initiative since its beginnings in 2003.


later in the web article, confesses that he didn’t actually write the Science paper, but rather uses it as an exam-ple of a disturbing trend in scientific publication.

I confess that I have my own “I confess” story. I confess I wrote a pa-per in 1992 describing the sequenc-ing of a tiny fragment of DNA from a termite entombed in thirty million year-old amber. The paper was pub-lished in Science and also gained a lot of public attention. While I still stand by this work, subsequent attempts to isolate DNA from amber preserved

specimens have failed miserably. The ancient DNA community stepped back and started to demand higher standards for ancient DNA work, and now the oldest specimen that has yielded DNA that the research community accepts is only 700,000 years old. The reason I am dredging up the arsenic DNA and amber in-sect stories is that they demonstrate a curious aspect of publishing and of public perception. After publish-ing the original Science paper, several better controlled experiments were done on the amber insect specimen

While doing a literature search for a manuscript recently, I came across an interesting web blog from Michael Eisen, a geneticist at UC Berkeley and a Howard Hughes Investigator. The article started with “I confess, I wrote the arsenic DNA paper.” For those of you not familiar with this story, the paper was published in Sci-ence in 2011, describing the discov-ery of a bacterial species that used arsenic instead of phosphorus in its nucleic acids. This bizarre finding received tons of press, but alas was shown to be incorrect. Dr. Eisen,

Science, Plain and BeautifulDNA barcoding may not be “sexy,” but it is beautiful in its simplicity and expediency.By roB DeSalle

Image: S.W

. Anderson


biodiversity. In the process, speci-mens are collected, archived and catalogued, producing an invaluable resource in and of itself. The barcode sequence is “icing on the cake.”

DNA barcoders, though, have dis-covered an excellent way to dissemi-nate the information and idea behind the endeavor. They use the approach to teach kids about biodiversity, ge-netics and biology. Because of its in-herent simplicity and low cost, the approach is amenable to dissemina-tion in this way. Cold Spring Harbor Laboratories and their DNA Learn-ing Center have developed a program called the Urban Barcoding Project. The UBP, initially supported by the Sloan Foundation and currently sup-ported by the Pinkerton Foundation, enlists high school students from the New York City area to characterize a group of organisms using barcoding. During the process, students learn a spectrum of basic biological princi-ples and techniques. The high school kids take on projects covering every-thing from the basic biology of speci-men identification to DNA sequenc-ing and informatics. It is a wonderful example of citizen science. I attended the first annual student awards com-petition, and the number of lit light-bulbs above the kids’ heads was stun-ning. To these kids, DNA barcoding and biodiversity was incredibly sexy.

Eleanor Roosevelt once said: “No matter how plain a woman may be, if truth and honesty are written across her face, she will be beautiful.” And so goes science. Plain, basic, in-the-trenches science like DNA barcod-ing is important and beautiful, and it needs to be made more accessible to the general public. nnn

Rob DeSalle, PhD, is a Curator and Pro-fessor at the American Museum of Natu-ral History and The Sackler Institute for Comparative Genomics.

founded in 1937, and over the past seventy-five years this institute has funded an enormous number of pro-posals, many of them peripheral to cancer, but all of them informative to basic science. Yet another example is the basic genetic work that conserva-tion biologists have accomplished in the last two decades characterizing variability in endangered and threat-ened species. While this work has aided in conservation policy, much of it goes unnoticed by the public.

So, short of scientists hiring pub-licity agents (some do, while oth-ers have strong public information departments at their institutions), what is a scientist to do? One strat-egy is to keep chasing “sexy” stories. A sexy story will almost always make it into a high visibility journal. The problem is, what is considered sexy is up to the expertise of the editors at the high visibility journals, as Mi-chael Eisen points out. A more sure way to accomplish this is to play the “broader impacts game” and put seri-ous thought into how to disseminate information to the public.

A somewhat neglected scientific endeavor in the “hidden” category of organismal biology is DNA barcod-ing, the topic discussed in this issue of GeneWatch. It makes sound basic science sense to catalog organisms. Sir Robert May once said: “Without taxonomy to give shape to the bricks, and systematics to tell us how to put them together, the house of biological science is a meaningless jumble.” This quote points to the essential impor-tance of cataloguing biodiversity. The problem is, cataloguing organisms does not sound “sexy” to the pub-lic (unless of course the Loch Ness Monster is actually found, named, archived and catalogued). DNA bar-coding wants to obtain a universal sequence from as many organisms as possible as a means to cataloguing

and these were published in lower visibility journals with no fanfare. I consider these subsequent papers orders of magnitude better than the original Science paper. Many scien-tists who have published in Nature or Science (the two preeminent science journals worldwide) will more than likely not point to their publications in those journals as their best science (but they will list them prominently on their CVs).

Most scientists would agree that getting the public to understand our work is very difficult and frus-trating. But it is perhaps one of the most important things we can do as scientists. And given the current at-titude many United States citizens have toward science, it has become essential that the public understand science better. Granting agencies like the National Science Foundation re-quire that a proposal have what is called a “Broader Impacts” section. These broader impacts are ways that researchers propose to disseminate their research to the general public. Even when the research is conducted at a museum (as mine is), it is still very difficult to push scientific work out into the public. Unless it is in-credibly “sexy,” the news media usu-ally ignores it. This means that many highly worthwhile scientific endeav-ors get overlooked or are invisible to the general public.

One example of “hidden” basic science is the hard basic work that virologists have done in trying to un-derstand infectious entities like HIV and HPV. The public more than likely is not interested in how these viruses replicate or how their genes are ex-pressed, yet these subjects comprise the scientific background for medical advances against the viruses. Anoth-er example is the basic science that grew up around research on cancer. The National Cancer Institute was


the forefront of research, the fact that anyone can take advantage of it by sending samples off to a laboratory meant the kind of investigative tools once restricted to Ph.D.’s and crime labs can move into the hands of cu-rious diners and amateur scientists everywhere.”

Readers of GeneWatch are prob-ably more aware than most of the as-tounding rate at which DNA science in general is progressing. What they may not know is that there is a grow-ing movement to democratize the technology, to put it into the hands of the public for the greater good. Pro-fessional scientists like myself have been inspired to found open, public-serving laboratories that are acces-sible to anyone who wants to pursue a safe and useful project. Genspace, which I co-founded and direct, is a

nonprofit community biolab located in Brooklyn, NY. We provide work-space, access to equipment, and mentorship in the biosciences. Gens-pace offers adult education courses, free public events such as open bar-coding nights, low-cost lab space for inventors, and is a place for students to work on projects for science com-petitions. One of the best uses of community labs is the kind of DIY investigation that can tell you more about your environment, health, or food. Is that goat cheese made with cow’s milk? Bring it in and we’ll teach you to barcode it. Want to know if your soy milk is Roundup Ready? We can teach you to determine that too, it’s an even simpler protocol than barcoding. We want everyone to be-come more literate in the biosciences in order to join the discussion about

In 2008, I happened to see an in-triguing news story about two New York City high school girls who had used a new DNA-based identifica-tion method to determine if their neighborhood sushi restaurants were selling mislabeled fish. That was my first encounter with the technique known as DNA barcoding. Since then, I have helped hundreds of ama-teur scientists use barcoding to ques-tion the identity of everything from ‘heirloom’ oranges to ‘beef ’ meatballs to the diversity of Alaskan plants.

The idea of identifying species through a very short genetic se-quence, rather like the manner in which a supermarket barcode identi-fies products, was first proposed in a 2003 paper by Dr. Paul Hebert, a re-searcher at the University of Guelph in Ontario, Canada. The beauty of barcoding is that even non-special-ists can obtain barcodes from tiny amounts of tissue and conclusively identify a species. Compare this to standard taxonomic identification, which requires intact specimens (of-ten impossible in situations where you want to know the identity of foodstuffs) and an expert able to dis-tinguish subtle anatomical differenc-es between closely-related species using morphological features like the shape and color of the organism’s parts. As the New York Times put it in their article about the abovemen-tioned ‘SushiGate’ kids:

“What may be most impressive about the experiment is the ease with which the students accomplished it. Although the testing technique is at

Citizen ScienceEasy enough for kids and amateur scientists to use, you might say DNA barcoding is inherently democratic.By ellen JorGenSen


necessary to get enough material in the tube to send out for sequencing.

PCR is a standard lab technique that has become mostly automated. Prepackaged mixtures of enzymes and reaction components such as the PCR primers that target the barcod-ing region can be bought cheaply in bulk. All one has to do is add a minute quantity of your DNA to the PCR mix and stick it into a preprogrammed machine. What comes out is ready to be sent off for sequencing at a fee-for-service facility doing hundreds of sequencing reactions daily. The total cost for the whole procedure can be less than $20 per sample.

Our barcoding nights have been very popular. They educate people and make them more informed about cutting-edge science. There is also a social component to the proj-ect where participants often engage in discussions about the promise and the repercussions of the technology.

It wasn’t that long ago that major scientific contributions were made by curious amateurs, and science it-self was less of a profession and more of a hobby. The popularity of our barcoding nights might be predictive of the resurgence of such citizen sci-ence, where a diverse cross-section of the general population are en-thusiastic participants in scientific inquiry. It’s empowering to be able to use the latest breakthroughs to answer questions of importance to you. I can think of no better use of my time than to continue to facilitate this empowerment through my work at Genspace. And please do stop by and barcode something if you are in the neighborhood! nnn

Ellen Jorgensen, PhD, is co-founder and President of Genspace, where she spearheads the Urban Barcode Project and other programs. She was an invited speaker at TEDGlobal 2012.

in the Alaskan landscape, where gla-ciers recede practically before our eyes and environmentalists attempt to preserve species diversity in the face of opposing economic interests. In Genspace’s Alaska Barcode Proj-ect, we invite the general public to monthly open nights where we teach them to barcode plant samples col-lected from remote locations in in-terior Alaska. The goal is twofold: to create a baseline survey of plants in particular areas such as the Skolai Valley in Wrangell-St. Elias National Park, and to add new identifying bar-codes to the Barcode of Life Database to empower future amateur scientists to conduct similar surveys.

Part of the DNA sequence of the chloroplast gene rbcL has been designated as one of the two barcode regions for plants (the matK gene is the other region but is not used at Genspace). Barcoding a specimen starts with extract-ing its DNA. You only need a small piece, the diameter of a

pencil eraser, to get plenty of DNA for barcoding. In a tiny plastic tube, the sample is mixed with a few drops of a solution that disrupts the cellu-lar structure and then ground into a paste using a little plastic pestle. The DNA is then absorbed onto silica, which is washed with salt-containing buffers until all other cellular com-ponents are gone. The clean DNA is eluted off the silica with water and the barcoding region amplified using a procedure called polymerase chain reaction (PCR). The amplification is

them from a position of knowledge as opposed to forming opinions based on ignorance and fear. And I strongly feel that the best way to learn is hands-on in the lab.

Barcoding is a regular activity at Genspace. It’s a great way for ama-teurs to participate in real science. Although the DNA barcodes of most common species have been depos-ited into public databases, most of the millions of species on earth have not been barcoded yet. This gives the student or citizen scientist an oppor-tunity to contribute to the growing public database of DNA barcodes. Genspace first began teaching bar-coding as part of Cold Spring Harbor Laboratory’s 2011-2012 Urban Bar-

code Project, a science competition for high school students. Genspace worked closely with their Harlem DNA Lab and acted as its satellite site in Brooklyn for teacher training and open lab hours to mentor students in barcoding, a relationship that contin-ues today.

Our newest barcoding project focuses on the importance of iden-tifying organisms to help monitor the biological effects of global cli-mate change. Accelerating habitat destruction is particularly evident


really all beef? Is this meat from an endangered species or illegally imported?

These are all critical questions that need to be answered on a daily basis by regulatory, commercial and enforcement entities around the world. DNA barcoding uniquely of-fers a single, standardized approach to this problem, yet it is not utilized broadly. Instead a variety of manual, time-consuming and expensive ap-proaches are used.

The obvious question: Why is DNA barcoding not used broadly outside the academic community if it offers such benefits in speed, accu-racy and cost in so many arenas?

The answer is two parts: One fac-tor is that many of the non-academic (applied) applications are governed by a legal umbrella that must be sat-isfied. The second important factor is that in these applied areas there are stakeholders, often with conflict-ing interests, that must be accounted for. The net result is that for barcod-ing approaches to be accepted and utilized broadly they must be imple-mented in a manner that is credible and satisfies the various stakeholders involved.

One example is water quality test-ing. Currently, a commonly used measure of water quality is to take samples from a water reservoir, such as a river, and measure the frequency of different species that are typically found in that habitat. As different species have different tolerances for

low water quality, the species profile is an excellent indicator of the quality of the water and possible pollution. There are well-established indices, based on extensive historical data, that were created using manual col-lection and taxonomic classification of species. Replacing those manual methods with DNA barcoding would greatly streamline the process, and making the switch seems like an easy decision.

However there are several stake-holders in this process who need to be satisfied. The regulators who use this data to monitor water qual-ity need to be assured that the new methodology is sound. The entities subject to the regulators oversight (local authorities, companies that discharge into the reservoir) need to accept the new methodology as fair and robust, and need to be assured that it does not represent a change in standard against which they are measured. There may also be legal requirements written into local law that need to be satisfied. To imple-ment a change in methodology from traditional approaches requires mul-tiple development activities: stan-dardized techniques for sample col-lection, storage and transport need to be developed and tested to ensure the integrity of the DNA is reliably maintained; standardized primer sets and DNA barcoding protocols need to be developed and tested; a DNA barcode database that reflects the local species profile needs to be

DNA barcoding is an immensely powerful technology. It offers the ability to take any biological sample and with a relatively fast, cost-effec-tive and accurate test, answer the simple question: What species is this?

The capability to answer the spe-cies ID question in a simple manner is valuable in many arenas.

• Customs and quarantine inspec-tors are interested in identifying biological materials they intercept at borders: Is this piece of meat from an endangered species? Is this larva from an insect that will devastate our national citrus har-vest? Is this wood illegally logged?

• Authorities responsible for moni-toring water quality are interested in identifying species present in rivers, streams and lakes: Does the species profile indicate that the water is polluted or pure?

• Companies and regulators in-volved in the medicinal plant business are interested in identi-fying plants used as raw materials: Is this dried plant I received from my supplier what he says it is? Is this plant material I am testing for a new product formulation what I think it is? Is this company label-ing its product accurately?

• Authorities responsible for moni-toring the integrity of the food supply need to have robust spe-cies identification methods: Is the fish being sold what the ven-dor says it is? Is this minced beef

What’s the Holdup?Outside academia – and especially in highly regulated environments – DNA barcoding is only gradually replacing slower, less accurate, and more expensive methods. Which begs the question: Why?By Peter chriStey


to perform such tests. 6. Optimization to meet local con-

ditions: One size does not fit all! A robust and acceptable barcod-ing system for use in one country, city or jurisdiction cannot simply be cloned across the globe – mod-ification and development to ad-dress local needs will be required.

The conditions outlined above are not unique to DNA barcoding. Two other examples offer models for what may be required for new tech-nologies to expand to widespread use outside academia. The first is the use of DNA-based technologies for hu-man identification in the forensic/law enforcement arena. The environ-ment for use of DNA technology for forensics has evolved over 20 years. Now in the U.S. there is a sophisticat-ed system of controls to ensure the integrity of the system. Before a new DNA-based product can be used in this environment, the manufacturer must perform comprehensive vali-dations to establish the performance parameters of the product. Guide-lines for these validations are provid-ed by an independent expert group. The product must be approved by the appropriate agencies before it can be used in conjunction with the national DNA profile database. Only certified labs may upload profiles into the da-tabase or search against the database. The database itself is subject to strict controls and regulations to ensure its integrity.

A second model is the in vitro di-agnostic model. Again we see simi-lar themes. Before a new diagnostic test can be used, its protocol must be standardized and validated. The test must go through clinical trials to prove its utility and performance. A governing body must approve the test. The test can only be utilized in appropriately qualified laboratories.

chain of custody and barcoding, may need to be established and validated. Standardization is of-ten facilitated by the availability of commercial products manufac-tured to rigorous standards under modern quality control systems.

2. Comparison to current meth-ods: In regulated environments, a full understanding of how bar-coding methodologies compare to current methodologies needs to be established.

3. Demonstration of benefits: The benefit of moving to barcode-based methodologies needs to be accepted.

4. Availability of a reliable refer-ence database: A robustly devel-oped and managed reference da-tabase for DNA barcodes needs to be developed. The publicly available databases represent an extraordinarily valuable resource for the scientific community. As content is derived from many sources with differing degrees of reliability, these databases are unfortunately not acceptable for use in more regulated or legalistic contexts.

5. Certification: A means to deter-mine that a laboratory is qualified

developed with controls and rigor that satisfies both the regulator and the regulated entities; studies dem-onstrating equivalence of results obtained from DNA barcoding vs. traditional approaches need to be performed. Funding this work, per-forming the various studies, dem-onstrating the benefit of change and gaining acceptance of the new meth-odology can take several years.

A second example is the use of DNA barcoding for monitoring la-beling of seafood in the U.S. The FDA facilitates programs related to the integrity of the U.S. seafood supply and have validated DNA barcoding for seafood identification. The FDA developed their own protocols and performed comprehensive valida-tions of the methodology. They also have developed their own DNA bar-code reference database under rigor-ous controls that satisfy the require-ments of the environment in which they work.1

It’s easy to assume that the FDA methods are easily portable to anoth-er country. However, while the FDA has established an excellent example of forward-thinking with a new tech-nology, another country establishing this technology for fish surveillance would need to do its own validations and testing to satisfy its own legal/regulatory environment and stake-holders. It would also need to devel-op its own DNA barcode database to reflect the idiosyncrasies of the local fish supply.

From these two examples we start to see why there are multiple steps that must be implemented before DNA barcoding can be used in regu-latory or legal contexts:

1. Standardization: Standardized protocols for key parts of the pro-cess, including sample collection, sample transport, sample tracking,


Again, similar systems governing in vitro diagnostics exist in different countries, but each has evolved dif-ferently to meet local conditions.

So, does this mean that it will be a long time before we see the wide-spread use of DNA barcoding tech-nology? Fortunately the answer is no. The requirements above apply to highly regulated and/or legalistic environments. Barcoding is a power-ful tool that will be applied in many areas where the requirements are not so high such as in educational pro-grams, national park management, biodiversity monitoring and envi-ronmental impact assessment. Even in more stringent environments, bar-coding may be used as a tool to guide investigations (but not used as evi-dence) and as a means of performing research.

Now that the scientific foundation of barcoding has been firmly estab-lished in the academic community, we are seeing many creative uses of this technology. Use will grow as more people become familiar with the technology and DNA barcoding becomes more accessible through the development of commercial products and services and less com-plex instrumentation. The formal processes and structures required for regulated/legalistic environ-ments will evolve in tandem – as for the biological ecosystems that DNA barcoding helps us understand, the DNA barcoding ecosystem itself will evolve and grow over time. nnn

Peter Christey, PhD, is Vice President and General Manager for the capillary electrophoresis and 5500 DNA sequenc-ing businesses at Life Technologies, Inc.

Consumers today are faced with almost daily risks to their ge-

netic privacy. A tsunami of personal genetic data is being cre-ated as genetic testing increasingly becomes an integral part of medical research and health care. The vast amount of genetic data being generated raises serious medical privacy concerns. Many Californians are afraid that their genetic information will be used against them and are unwilling to participate in medical research or to be tested clinically, even when they are at substantial risk for serious disease. The public simply does not trust insurers, em-ployers and other entities with incentives to improperly acquire and use genetic information.

Despite the passage of several new laws to protect genetic pri-vacy, many remain unaware of their privacy rights, of where they are protected and where they aren’t. It’s not difficult to ascer-tain why: there has never been a comprehensive public education program on genetic privacy.

That is why the Council for Responsible Genetics and the Al-liance for Human Biotechnology are pleased to announce the creation of the California Genetic Privacy Network; an ongoing project to educate Californians and the greater public about ge-netic privacy rights.

The Network’s website will serve as a resource for Californian patients, consumers and other front line actors to have an in-formed understanding of their genetic privacy rights under Cali-fornia and federal law. The California Genetic Privacy Network also offers in person and online educational consultations.

Check out the California Genetic Privacy Network website today at:

www.geneticprivacynetwork.org

Launch of the California

Genetic Privacy Network

Information, Guidance, and Training on

California Genetic Privacy Protections


in the Smithsonian’s National Muse-um of Natural History in September 2004 with the mission to support the development of DNA barcoding as a global standard for species identifi-cation. CBOL took on an ambitious program of work to build a coherent, collaborative community of practice, including:

• Creating the BARCODE data standard for high-quality data re-cords in GenBank, the European Nucleotide Archive and the DNA Data Bank of Japan;

• Convening Working Groups that would conduct research leading to selection of the standard bar-code region for plants, fungi and protists;

• Facilitating the launch of sev-eral major international barcod-ing ‘campaigns’ such as the All Birds Barcoding Initiative and FISH-BOL;

• Engaging the participation of de-veloping countries through out-reach workshops in southern, eastern and western Africa, Latin

America, east Asia, China and India;

• Representing DNA barcoding to international organizations such as the Convention on Biological Diver-sity (CBD), the Convention on the International Trade of Endangered Species (CITES), the Food and Ag-riculture Organization (FAO) and

Wildlife crime has emerged in recent years as one of the top four international crimes, representing tens of billions of dollars per year and ranking alongside smuggling of drugs, weapons and human slaves. As the price of products made from endangered and protected species has increased, organized crime net-works and terrorist organizations have entered this trans-boundary trade. These products include carved ivory, traditional medicines such as powdered rhino horn and lion bone, ‘bushmeat’ for consumption and re-ligious rituals, exotic pets, leather goods and rare plants valued by landscape designers. To evade de-tection and prosecution, smugglers have learned how to make it virtu-ally impossible to identify the species of origin by removing the diagnostic morphological features that experts can use to identify the contraband products. Only the DNA in the prod-uct can bear witness to the species of origin.

Shortly after DNA barcoding for animals was proposed in Prof. Paul Hebert’s 2003 publication, the Alfred P. Sloan Foundation of New York sponsored two workshops to develop a roadmap for the development of barcoding as a global research tool. An important part of the roadmap was the establishment of the Consor-tium for the Barcode of Life (CBOL) through Sloan Foundation support to the Smithsonian Institution. CBOL officially opened its Secretariat Office

The Barcode of Wildlife ProjectDNA barcoding takes center stage in the international effort to combat wildlife trafficking.By DaviD SchinDel

Photo courtesy of the Kenya Wildlife Service.


that partner country. CBOL has developed a project

roadmap with four phases:

1. Planning and assessment of part-ner country capabilities for DNA research and forensic science, ex-ploration of legal standards for use of wildlife DNA in prosecution, and selection of priority endan-gered species. The four participat-ing partner countries have com-pleted this phase and the priority species on which they will focus can be seen on the Endangered Species Viewer;

2. Training of researchers, enforce-ment officials, forensic lab techni-cians, prosecutors and others;

3. Construction of reference DNA barcode library and testing of fo-rensic lab capabilities to identify species with DNA barcodes; and

4. Implementation, investigation of crimes using DNA barcodes, and prosecutions.

The project’s goal is to prepare and empower each partner country to conduct barcoding within its bor-ders, without relying on the export of any biological samples to foreign labs. Simply put, BWP wants to im-port technology and capability, not to export specimens. Over the course of the project, partner countries will receive support for:

• Specimen sourcing: CBOL will coordinate construction of the barcode reference library which will be based on voucher speci-mens from around the world. This budget category covers the cost of technician salary for tissue sub-sampling, shipment of samples to processing labs, honoraria for tax-onomists who will verify species identifications, and management of data and metadata.

• Processing specimens for

In July 2012, CBOL was ap-proached by Google Giving with the opportunity to submit a project pro-posal involving species protection. In October 2012, the Smithsonian re-ceived a US$3 million Global Impact Award Barcode to CBOL for the Bar-code of Wildlife Project (BWP). The project is structured as a partnership between CBOL and six partner coun-tries. South Africa, Kenya, Nigeria and Mexico began participating im-mediately and countries in Asia and South America will be selected soon to fill the remaining slots.

BWP has three goals:• Create a high-quality public refer-

ence database with standardized ‘DNA barcodes’ for the endan-gered species that are the high-est priority to the partner coun-tries. The project hopes to include 50,000 data records from 10,000 species, including 2,000 protected species and 8,000 closely related and look-alike species;

• Enable each partner countries to identify crime exhibits by com-paring their DNA barcodes with the reference library; and

• Support efforts by partner coun-tries to adopt DNA barcoding as a standard, sustainable tool for the investigation and prosecution of wildlife crime.

Each partner country assembles a National Project Committee (NPC) of about 10 representatives of en-forcement agencies, prosecutors, and academic researchers in biodi-versity science. This mix of people is critical because the project’s suc-cess relies on participation of both the providers and users of barcode data. Each NPC is co-chaired by an enforcement official and a leading biodiversity research scientist from

the Convention on Phytosanitary Measures (CPM);

• Creating a social network for the barcoding community and an in-formational website to explain barcoding to diverse audiences;

• Promoting the adoption of bar-coding by US governmental agen-cies such as the Department of Agriculture, Environmental Pro-tection Agency, Food and Drug Administration, and the National Oceanic and Atmospheric Ad-ministration; and

• Organizing international barcod-ing conferences held in London (2005), Taiwan (2007), Mexico City (2009) and Adelaide, South Australia (2011).

At the Adelaide conference, CBOL’s Executive Committee decid-ed that CBOL’s original mission had been accomplished to a great degree but a new challenge had emerged. Government agencies and private companies had not yet started to for-mally adopt and invest in barcoding. The problem seemed to reside in the lack of a public reference database of sufficient reliability. Despite the success of the BARCODE data stan-dard in GenBank, barcoding was still viewed as a research tool for taxono-my, not the basis for regulatory or le-gal affairs. The Executive Committee decided that CBOL should become directly involved in a large and ambi-tious project to meet and overcome this challenge in one of five areas of application:

• Protecting endangered species,• Enforcing truthful labeling of food

in the marketplace,• Testing water quality,• Regulating labeling of medicinal

plants and herbal remedies, and • Preventing the international in-

troduction of agricultural pest species.


David Schindel, PhD, is the Executive Secretary of the Consortium for the Bar-code of Life, a project hosted by the Smith-sonian Institution’s Museum of Natural History.

• Information about the Consor-tium for the Barcode of Life: http://www.barcodeoflife.org/content/about/what-cbol

nnn

construction of the reference barcode library and forensic ap-plication: Specimen processing will be done in-country for some partner countries but others with-out molecular labs will need to ex-port samples, at least initially. This amount will cover lab technician salary, reagents and consumables and data management. Funds are not available for lab facilities or equipment, though CBOL is will-ing to work with partner countries to seek additional funding for ca-pacity-building. Partner countries will be expected to provide access to laboratory facilities during the project. If the project proves suc-cessful, CBOL and Google hope that partner countries will make barcoding a normal part of their budgets for CITES enforcement after the grant ends.

• Planning, training, and out-reach: This category includes the costs of planning meetings, train-ing of technicians, researchers and enforcement officials, outreach to related organizations such as the CITES Secretariat, IUCN, Inter-pol, the Convention on Biological Diversity, and potential donors. Partner countries will be expected to cover staff salaries for people receiving training, and the costs related to the work of the National Project Committee between plan-ning meetings in which CBOL is directly involved.

The following websites provide useful background information about CBOL and BWP:

• Information about the Barcode of Wildlife Project: www.barcodeof-wildlife.org

• Information about DNA barcod-ing: www.barcodeoflife.org

Photo courtesy of the Kenya Wildlife Service.


successfully been used in the iden-tification of species pursuant to a criminal investigation. However, the scope of its application has been very narrow, due to the significant cost and time implications to un-dertake this analysis; as well as the limited number of species for which these tests could produce effective results.

The ability to quickly, consis-tently and accurately identify suspi-cious environmental commodities has always been a stumbling block for Environmental Management Inspectors, preventing them from entering the next critical phase of criminal investigations. The DNA Barcoding Project has the ability to provide the green light for these law enforcement officials to proceed.

Examples of its application to en-vironmental contraband include:

• Cycads that have been stripped of their leaves and roots being smuggled from the wild to plush suburban homes;

• Endangered plants that are ei-ther ground into a powder or from which oils are extracted;

• Look-alike species that are smuggled under the protection of a permit;

• Lion bones that have been cooked and cleaned.

It is hoped that the coming to-gether of the scientific, academic and law enforcement disciplines

in the DNA Barcoding Project will keep our Environmental Manage-ment Inspectors one step ahead of offenders — and the criminals on their toes. nnn

Jacques du Toit is Deputy-Director of EMI Capacity Development at South Africa’s Department of Environmental Affairs.

“As fast as one thing evolves, an-other evolves to keep up with it…” This quotation aptly describes the “cat and mouse” relationship be-tween environmental law enforce-ment officials and criminals whose activities threaten the survival of so many endangered species. As law enforcement (driven by the need to conserve our natural re-sources for the benefit of future generations) improves its networks, strategies and technology to effec-tively tackle contraventions of en-vironmental laws, so too does the criminal element adapt to changing circumstances, allowing them to pursue their objective of personal enrichment.

In South Africa, one of the evo-lutionary steps that has the poten-tial to swing the pendulum in favor of environmental law enforcement (represented by the Environmen-tal Management Inspectorate) is the application of DNA barcod-ing in the environmental criminal investigative and forensic field. By no means a new science within the academic world, the application of DNA barcoding is set to become a “a first step on the moon” moment for Environmental Management Inspectors hungrily awaiting its conversion into a practical law en-forcement tool that will allow them to extend their reach to criminals previously beyond the scope of identification.

The use of DNA analysis has

Foiling Poachers With DNA BarcodingIn South Africa, where poachers and smugglers continually adapt to stay a step ahead of environmental law enforcement, DNA barcoding is about to be a game changer.By JacqueS Du toit

Lion paw bones. Image provided by the Department of Environmental Affairs, South Africa.


was restricted by the cost and techni-cal skill required to perform the anal-yses. As a result, it was not typically utilized during regulatory investiga-tions. In 2003, a series of publications by a research group at the University of Guelph in Ontario Canada first highlighted the concept of “DNA Barcoding” for the FDA. This use of short fragments of DNA, generated

and analyzed under a standardized set of conditions, seemed like an ideal alternative to the protein based meth-ods of the day. Advances in sequenc-ing technology and techniques, which reduced both the cost and expertise required to perform these tests, al-lowed the FDA to modernize many of its food analysis laboratories around the country to include DNA testing. Working with many of the pioneers of DNA Barcoding, including the

Canadian Center for DNA Barcoding and the Smithsonian Institution, the FDA has developed a standardized protocol for DNA Barcode generation allowing the Agency to fully replace the older protein based methods in its regulatory investigations.

An early example of FDA’s use of DNA analysis of seafood was during a 2007 investigation of several food-

borne illness outbreaks in Illinois, California, and New Jersey. The illnesses were linked to fish that had been illegally imported into the U.S. from China as “headless monkfish” and sold in several Korean retail establishments and restaurants as “Bok.” DNA analysis revealed that the fish were in fact a species of pufferfish that was not al-lowed for import. The meat of that particular species of fish is known to be highly contaminated with the puff-erfish toxin tetrodotoxin, making safe preparation of the species impossible. DNA barcoding is now used regu-larly to confirm the species of seafood associated with

foodborne illness outbreaks. This has already resulted in a better un-derstanding of food safety hazards associated with specific species of seafood, which in turn helps FDA to further refine its guidance for con-trolling these hazards and hopefully prevent outbreaks from occurring.

Use of this technology need not be limited to regulatory officials. Prop-er species identification by the sea-food processing industry is essential

DNA sequencing provides public health officials with an important tool which can be used to help ensure that seafood products are safe and prop-erly labeled. When the FDA is investi-gating either a foodborne illness out-break linked to a seafood product or a seafood mislabeling case, the Agency wants to have a high degree of cer-tainty when confirming the identity of the product in question – no small task, given that there are over 1,700 species of fish and shellfish poten-tially found in the U.S. mar-ketplace. In almost all cases, DNA sequencing can pro-vide that certainty. The ben-efits of using DNA to iden-tify species aren’t just limited to public health officials; it can be an invaluable tool for industry when it comes to ensuring that the proper food safety measures are in place during processing and that the product is properly labeled.

Historically, regulatory seafood species identifica-tions either relied on visual examination of the external characteristics of the product, which are often removed during process-ing, or crude protein profiling meth-ods that were sometimes difficult to interpret or did not work on heav-ily processed products due to protein degradation. Despite its limitations, protein based seafood identification methods were the regulatory stan-dard for many years. Techniques in-volving DNA were sometimes used in high profile cases but its routine use

Identifying Commercial Seafood DNA barcoding is a valuable tool for FDA for the species identification of seafood.By Jonathan DeeDS


because the controls necessary to en-sure the safety of seafood during pro-cessing are determined by the species of seafood being processed. Proper labeling of seafood is also dependent on knowing the species of seafood. To assist the seafood industry in label-ing their products in a manner that is truthful and not misleading, FDA has published a guidance document called “The Seafood List.” It contains a list of seafood species potentially found in U.S. commerce, the accept-able market names for each species, and guidance for developing accept-able market names for new species. With the increased globalization of the seafood trade, new species are al-ways being introduced into the U.S. marketplace. If there is ever any ques-tion as to the identity of the fish a pro-cessor receives, DNA sequencing can provide the answer and help ensure that it is safely processed and properly labeled.

As the technology progresses and sequencing equipment becomes smaller, more affordable, and more transportable, its use by regulators and industry members will likely in-crease further. The ability to perform this analysis outside of centralized laboratories and directly at the point of importation or processing will fur-ther ensure the safety and accurate labeling of seafood in the U.S. nnn

Jonathan Deeds, PhD, is a Research Bi-ologist at the U.S. FDA Center for Food Safety and Applied Nutrition.

able to collect enough DNA from 91% of the herbal products to deter-mine which species were inside. They found that 59% of the products in-cluded DNA barcodes from species not listed on the label, and only two of the twelve companies had prod-ucts which didn’t include fillers, sub-stitutes, or contaminants.

Some of the examples are aston-ishing. In addition to the Echinacea and Gingko biloba examples above (yes, those were both real), the re-searchers found two products labeled St. John’s wort that contained no St. John’s wort (one instead contained Alexandrian senna, a laxative); cap-sules that were supposed to contain dandelion also included grass, or contained no dandelion and instead only wheat and something in the ba-nana family. Fully 9% of the products turned out to contain no trace of the herb listed on the label, only fillers and substitutes.

The University of Guelph labora-tory that conducted this research is working on standardized testing pro-cedures and a DNA barcode library for commercial herbal species which herbal supplement companies could use to accurately and inexpensively authenticate their products. Given the results so far, that might be-come standard practice in the herbal supplements industry sooner rather than later. nnn

See the full study at http://www.biomedcentral.com/ 1741-7015/11/222.

When you buy medicine, you probably don’t question whether it actually contains the active ingredi-ents listed on the bottle. If you live in one of the world’s more-developed countries, your government probably has a stringent enough drug approval process to prevent egregiously mis-labeled medications from reaching the shelves, or at least to pull them from the shelves if mislabeling is identified. But what if you take herb-al supplements? Who makes sure the ground-up leaves inside an Echina-cea pill are really Echinacea and not, for example, an invasive weed which has been known to cause allergies and skin rashes? If you have a nut al-lergy, how do you know your Ginkgo biloba doesn’t have, say, black walnut mixed in with it?

Herbal supplements are subject to much less regulatory oversight, and until recently, it would have been very difficult (and often practically impossible) to crack open a pill and determine which species were inside. But now, with the advent of DNA barcoding, researchers or regulators can do just that – and so far, it’s not looking good for herbals.

A study published in the journal BMC Medicine this fall used DNA barcoding to test the authenticity of 44 herbal supplements from 12 com-panies. To do this, the lab (at Univer-sity of Guelph in Canada) created an herbal barcode library for reference, then matched DNA barcodes from the supplements to those in the ref-erence library. The researchers were

Uncovering Herbal Fraud With DNA BarcodingHow do you know your Gingko biloba is Ginkgo biloba? A new study shows that’s a very valid question (and answers it).By crG Staff


Currently, the gold standard for mak-ing sure there’s no ruminant material in animal feeds is microscopy. You have a microscopist who actually looks at the particles in the feed to determine whether it contains ani-mal material. The problem with that is if you find, for example, a hair in the feed, it doesn’t tell you if this hair is from a cow, a sheep, or a pig. So al-though microscopy is the gold stan-dard, it has a very difficult time iden-tifying species. So you have to fall back on these molecular methods, because if there’s hair there, we need

legally sold – are they actually bear steaks?

I’m trying to imagine someone test-ing a piece of dry dog food. Where are the samples collected to make sure you have enough to get a DNA barcode?

We can extract DNA from really any material now. If it’s wet dog food, dry dog food … we’ve actually done a lot of work with pet jerky, too. It’s been on the news recently, there’s con-cern about dogs getting sick from pet jerky – we did the testing of the jerky to make sure it is what’s on the label. So really any material we have, we can extract DNA from.

But when we talk about animal feed that has been rendered, it can be very difficult to create a full-length bar-code. So my colleague Yolanda Jones is not only validating full-length bar-codes, but also looking at what they call “mini barcodes.” Whereas regu-lar barcodes are about 650 base pairs, minis are only 100 base pairs. The materials we look at have sometimes been degraded, whether by cooking or rendering or just sitting out on a dock somewhere, which makes it very difficult to create an amplified product of 650 base pairs, but you can still create a product of 100 base pairs and use the same approach and still identify what that species is.

If you weren’t using DNA barcod-ing, what’s the alternative? For ex-ample, how else would you identify ruminant material in feeds?

Haile Yancy, PhD, is a senior research bi-ologist at the U.S. Food and Drug Admin-istration’s Center for Veterinary Medicine.

GeneWatch: Can you tell me a bit about how DNA barcoding relates to your work at FDA? How was it first used for monitoring animal products or feeds?

Haile Yancy: We started out using it for preventing mad cow disease. The issue with mad cow disease is that ruminant material – bovine, sheep, or goat – is not allowed to be put into animal feeds and fed back to cows, because that’s how the disease spreads. So we didn’t use DNA bar-coding per se to develop those assays, but we used those DNA sequences that are unique in barcoding and designed species-specific primers which we use to make sure that no ruminant material is in animal feeds.

More recently, the Chicago office asked for our help identifying po-tentially mislabeled game meat and potentially mislabeled pet foods. These concerns are similar to the concerns with seafood mislabeling, where someone sells one product but labels it as something else so you can charge a higher price. With pet foods, there was concern about, for example, someone charging a higher price for gourmet duck pet food – was it actually duck meat?

It’s the same thing for the game meats. FDA has jurisdiction over game meats, and they wanted to make sure that if a company is sell-ing, let’s say, bear steaks – which are

Monitoring Animal Products and Feeds With DNA Barcoding

interview with haile yancy, u.S. fooD anD DruG aDminiStration


One thing we’ve been working on is concerns about heparin (an anti-coagulant) being made from non-porcine material, specifically from cow material. The method we’ve de-veloped for testing this is actually a modification of our assay for finding bovine material in feeds. That’s the safety aspect, but there’s also a reli-gious aspect, because if you don’t eat pigs you’re concerned about heparin being made from porcine material, so you want the opposite – you want to make sure your heparin products are derived from cow rather than pig material.

I see a lot of other uses for this for everyday things that we take for granted. For instance, if you’ve read the papers, there have been issues with food supplements being mis-labeled. I can see DNA barcoding being used more and more as two things happen: Firstly, developing more efficient ways of extracting DNA and being able to sequence it; and secondly, making sure that the cost goes down. As those happen, you’ll see a large application. And consumers are becoming more and more aware of things being substi-tuted and mislabeled – like the gro-cery store that found out some of the meat it was selling was not beef, but horse. More and more consumers are asking: Am I buying what I think I’m buying? And it matters from both an economic and a safety point of view. It’s way off in the future, but one of the long-term things we’re trying to work on, with Barcode of Life, is a handheld device that can do all these things. We’re heading in that direction. nnn

That’s pretty expansive!

Yes, it’s a fairly new group of individ-uals, but we’ve got the wind behind our back with the presidential ex-ecutive order. Some of these diseases are very nasty, and it’s something we hope can be prevented with all these agencies working together. We’re working very quickly to develop a method of barcoding so that when someone confiscates material at the border, they will be able to know what it is and make sure the risk is minimized.

Are you using existing reference databases for this, like the Barcode of Life?

Yes – one of the things we recog-nized very early is that it would be virtually impossible for the FDA by themselves to get the materials to build these reference databases. I’ve been working with Barcode of Life for over 12 years now. Their goal is to prevent the illegal importation of endangered species, so what we’ve done is piggyback on that, because if you look at the list for bushmeats and the list for endangered species, there is an overlap of common species. We don’t have the infrastructure to go to Africa and actually get samples of li-ons and elephants, so we have access to their specimens to help populate our database.

Do you see any new uses for DNA barcoding in the pipeline or farther down the road?

Because the technology is there to determine species of origin, you can see a shift in making sure, both on the producer and consumer side, that consumers get what they pay for – not only for economic reasons, but also for safety.

to determine where it’s from. That’s what’s important: Not just determin-ing whether there’s animal material in the feed, but is this material from a ruminant species?

Are there other uses of DNA bar-coding at FDA currently in use or planned besides the ones you have mentioned?

We recently had a group that was looking at prevention of infectious diseases transmitted to humans by bushmeat. People had been bring-ing back exotic meats, or bushmeat, from other countries. There’s an ex-ecutive order that the President just signed to combat wildlife trafficking, and one of the ways we’re doing that is putting together a national smug-gling prevention network to prevent the illegal importation of bushmeat into the United States. Part of that effort is to identify which species are brought into the U.S. as bushmeat, so part of what FDA has been tasked with is creating a DNA barcode da-tabase to use for this. If you have a piece of meat come in, it may not have the distinguishing characteris-tics that tell you “this is a monkey” or “this is an alligator;” so what we’ll do is barcode that, match it against the database, and once you figure out what kind of bushmeat that is, you can associate it with risk. Spe-cific bushmeats carry a specific risk – for example, primates can carry herpes virus or anthrax virus. It’s very difficult to establish risk with-out understanding which species it is. We’re part of a group working on this that includes the FDA, the U.S. Department of Agriculture, Customs and Border Protection, the Center for Disease Control, Fish and Wild-life Service, and hopefully including state and local governments.


for molecular work, DNA barcoding for bioassessment is comparable in cost or even cheaper than standard morphological methods.

3) Identifications based on DNA barcodes are at the species level, but morphological identifications, be-cause many benthic organisms are small or are juvenile life stages, are often at the family or genus level, especially for freshwater ecosystems

where accurate bioassessment is the most critical.

4) DNA sequence identifications are not subjective in that no matter how many times the same sequence is queried against the database, the identification will always be the same, whereas morphological IDs performed by different taxonomists or laboratories are known to raise

disagreements over IDs. 5) DNA-based identification can

be applied to all biota, including groups normally omitted from stan-dard bioassessment because of their small size (less than 0.5 mm) or ex-treme difficulty in identification (e.g nematodes).

Current and past EPA research into DNA barcoding has shown sev-eral interesting results. Our previ-

ous research has shown that DNA barcoding of benthic invertebrates at a large scale is feasible, and that the larg-est impediment to success-ful DNA work is in proper preservation of samples as opposed to any of the molec-ular genetic techniques. Our current collaborative work with research groups like the Southern California Coastal Water Research Project (SC-CWRP) and Stroud Water Research Center has helped develop new protocols for the collection and storage of benthic invertebrates for mo-lecular genetic workflows,

and has shown that DNA barcoding identification of freshwater benthic invertebrates provides more infor-mation than standard IDs and that this information has value for assess-ing environmental condition.

Ongoing research at EPA has moved into the use of Next Genera-tion DNA Sequencing for bulk pro-cessing of samples. The development

At the U.S. EPA, several projects in the Office of Research and Devel-opment have been investigating the use of DNA barcoding for identifying various aquatic organisms to species. One of the main areas of research is in determining the utility of DNA barcoding for environmental bioas-sessment — evaluating the environ-mental health and condition of a site based on the organisms found living there. Standard bioassess-ment relies on identifica-tion of biota, typically ben-thic invertebrates, using morphological characters. These morphological IDs are done by highly trained taxonomists, which can take many months or even a year to complete. Our work at EPA, along with collaborators outside the agency, seeks to capitalize on the potential speed and cost savings associated with identifying these ben-thic organisms through DNA barcode data.

The use of DNA bar-coding for environmental bioassess-ment potentially has several signifi-cant advantages:

1) Identification by high-through-put molecular techniques is consider-ably faster than current morphologi-cal techniques, with samples being processed in weeks to 1-2 months, as opposed to 6-18 months.

2) With continued cost declines

Measuring the Health of Aquatic Ecosystems With DNA BarcodingThe U.S. Environmental Protection Agency is finding DNA barcoding to be faster, more precise, and more accurate than previous methods of aquatic bioassessment.By erik PilGrim


genetic applications could be a trans-formative technology for the field of bioassessment. Our ultimate goal is to provide better information in a timely, cost-effective manner, in or-der to provide decision makers with best methods for assessing aquatic environmental health and condition. nnn

Erik Pilgrim, PhD, is a Research Scien-tist at the U.S. Environmental Protection Agency, where he applies DNA barcod-ing to bioassessment and invasive species monitoring and detection.

of Next Generation Sequencing techniques for environmental bioas-sessment has the potential to speed aquatic community identification and lower costs even more through DNA extraction, PCR, and DNA sequencing of bulk, unprocessed benthic samples. Current research investigates these NGS applications for stream, lake, and coastal marine samples. Our lab is working to de-velop techniques and workflows to generate the most useful data in the most cost-effective manner while also working to be user friendly for end users and decision makers. To-ward these goals, we continue to collaborate with non-agency re-search groups such as SCCWRP, as well as maintaining strong relation-ships with international research-ers in this field such as the Canadi-an Centre for DNA Barcoding and CSIRO–Australia.

Several challenges lie ahead for the development of DNA barcoding for bioassessment. Our first challenge is to foster better communication be-tween molecular genetic research-ers and environmental scientists, to ensure that the work is collaborative instead of confrontational. Second, the data generated through DNA-based identification is different from standard bioassessment, and molec-ular geneticists and environmental researchers need to work together to determine the most appropriate uses and analyses of this new data. Lastly, this molecular work will gen-erate much more data than has ever been possible for bioassessment, so methods must be developed to han-dle such large amounts of data while providing the most useful outputs for making determinations of environ-mental health.

This is an exciting time for both DNA barcoding and environmen-tal bioassessment. These molecular

From the Council for Responsible Genetics on the 30th Anniversary of GeneWatch magazine:

Biotechnology in Our LivesWhat Modern Genetics Can Tell You about Assisted Reproduction, Human Behavior, and Personalized Medicine,

and Much MoreEdited by Sheldon Krimsky and Jeremy Gruber

For a quarter of a century, the Council for Responsible Genetics has provided a unique historical lens into the modern history, science, ethics, and politics of genetic technologies. Since 1983 the Council has had leading scientists, activists, science writers, and public health advocates researching and reporting on a broad spectrum of issues, including genetically engineered foods, biological weapons, genetic privacy and discrimination, reproductive technologies, and human cloning. Written for the nonscientist, Biotechnology in Our Lives examines how these issues affect us daily —whether we realize it or not.

AVAILABLE NOWfrom Skyhorse Press

Stoeckle, p. 5

1. Hebert PDN, Cywinska A, Ball SL, et al (2003) Proc R Soc Lond B 270:313.

2. More specifically, those loci are: Animals, 5’ COI (2005); plants, rbcL/matK (2009); and fungi, ITS (2012).

3. Ratnasingham S, Hebert PDN (2013) PLoS ONE 8:e66213.

4. Newmaster SG, Grguric M, Shanmughanandhan D, et al. (2013) BMC Medicine 11:222

5. http://www.urbanbarcodeproject.org6. Santschi L, Hanner RH, Ratnasingham

S, et al (2013) PLoS Biol 11:e10014717. http://malaiseprogram.ca

Christey, p. 11

1. Further detail can be found on the FDA website at: http://www.fda.gov/Food/FoodScienceResearch/DNASeafoodIdentification/default.htms

Endnotes

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