DNA FISHES

DNA Hook - Students Find Bad Labels

2009-01-11T10:00:00.000-08:00

Fish Tale Has A DNA Hook - Students Find Bad Labels
From the New York Times - August 22, 2008
Many New York sushi restaurants and seafood markets are playing a game of bait and switch, say two high school students turned high-tech sleuths.
In a tale of teenagers, sushi and science, Kate Stoeckle and Louisa Strauss, who graduated this year from the Trinity School in Manhattan, took on a freelance science project in which they checked 60 samples of seafood using a simplified genetic fingerprinting technique to see whether the fish New Yorkers buy is what they think they are getting.
They found that one-fourth of the fish samples with identifiable DNA were mislabeled. A piece of sushi sold as the luxury treat white tuna turned out to be Mozambique tilapia, a much cheaper fish that is often raised by farming. Roe supposedly from flying fish was actually from smelt. Seven of nine samples that were called red snapper were mislabeled, and they turned out to be anything from Atlantic cod to Acadian redfish, an endangered species.
What may be most impressive about the experiment is the ease with which the students accomplished it. Although the testing technique is at 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 curious diners and amateur scientists everywhere.
The project began, appropriately, over dinner about a year ago. Ms. Stoeckle’s father, Mark, is a scientist and early proponent of the use of DNA bar coding, a technique that greatly simplifies the process of identifying species. Instead of sequencing the entire genome, bar coders — who have been developing their field only since 2003 — examine a single gene. Dr. Stoeckle’s specialty is birds, and he admits that he tends to talk shop at the dinner table.
One evening at a sushi restaurant, Ms. Stoeckle recalled asking her father, “Could you bar code sushi?”
Dr. Stoeckle replied, “Yeah, I think you could — and if you did that, I think you’d be the first ones.”
Ms. Stoeckle, who is now 19, was intrigued. She enlisted Ms. Strauss, who is now 18.
Their field technique was simple, Ms. Stoeckle said. “We ate a lot of sushi.”
Or, as Dr. Stoeckle put it, “It involved shopping and eating, in which they were already fluent.”
They hit 4 restaurants and 10 grocery stores in Manhattan. Once the samples were home, whether in doggie bags or shopping bags, they cut away a small piece and preserved it in alcohol. They sent those off to the University of Guelph in Ontario, where the Barcode of Life Database project began. A graduate student there, Eugene Wong, works on the Fish Barcode of Life (dubbed, inevitably, Fish-BOL) and agreed to do the genetic analysis. He compared the teenagers’ samples with the global library of 30,562 bar codes representing nearly 5,500 fish species. (Commercial labs will also perform the analysis for a fee.)
Three hundred dollars’ worth of meals later, the young researchers had their data back from Guelph: 2 of the 4 restaurants and 6 of the 10 grocery stores had sold mislabeled fish.
Dr. Stoeckle said he was excited to see a technology used in a new way. “The smaller and cheaper you make something,” he said, “the more uses it has.” He compared bar coding to another high-tech wonder turned everyday gadget, GPS.
Eventually, he predicted, the process will become more automatic, cheaper and smaller so that a handheld device could perform a quick analysis and connect to the database remotely. What his daughter did, he said, is like dropping film off at the supermarket for developing. The next generation could be more like a digital camera that displays the results on the spot.
The results of Ms. Strauss and Ms. Stoeckle’s research are being published in Pacific Fishing magazine, a publication for commercial fishermen. The sample size is too small to serve as an indictment of all New York fishmongers and restaurateurs, but the results are unlikely to be a mere statistical fluke.
The experiment does serve as a general caveat emptor for fish lovers, particularly because the students, their parents and their academic mentor all declined to give the names of the vendors, citing fear of lawsuits. Besides, they noted, mislabeling could occur at any stage of the process.
Dr. Stoeckle was willing to divulge the name of one fish market whose products were accurately labeled in the test: Leonards’ Seafood and Prime Meats on Third Avenue. John Leonard, the owner, said he was not surprised to find that his products passed the bar code test. “We go down and pick the fish out ourselves,” he said. “We know what we’re doing.” As for the technology, Mr. Leonard said, “it’s good for the public,” since “it would probably keep restaurateurs and owners of markets more on their toes.”
Ms. Stoeckle said the underlying message of the research was simple: “If you’re paying for white tuna and you’re eating tilapia, I think you’d want to know that.”
Although the students did not present the project for a grade at school, they made sure to mention it on their college applications. Both will enroll at Johns Hopkins University this fall.
Neither, however, expects to major in the sciences. “I’ve always been into art history,” Ms. Strauss said, “which is really different from this.” Ms. Stoeckle, who is the granddaughter of the entertainer and arts patron Kitty Carlisle Hart, is thinking about studying writing or psychology. But that, they said, is the point. “If we found it interesting — which we did — I think lots of people like us can do it, too,” Ms. Stoeckle said.
Peter B. Marko, a professor at Clemson University who used a more detailed genetic technique in a 2004 paper to show that red snapper was commonly mislabeled, called their project “quite remarkable,” though he added that genetic analysis had been simplified to the point that high school students could now perform the task without sending samples off.
Mr. Marko prefers to work with whole genomes — “more information is better,” he explained — which can be sequenced now with lightning speed. He plans to perform a broad genetic comparison of fishes that were separated millions of years ago by the rise of the Isthmus of Panama. “The technology is allowing us to ask questions that really would not have been possible in the past.”
The students worked under the tutelage of Jesse H. Ausubel of Rockefeller University, a champion of the DNA bar coding technique. As for Ms. Strauss and Ms. Stoeckle, Dr. Ausubel said they “have contributed to global science” by adding to the database, built on a model similar to that of Wikipedia, in which people around the world can contribute.
In a way, Dr. Ausubel said, their experiment is a return to an earlier era of scientific inquiry. “Three hundred years ago, science was less professionalized,” he said, and contributions were made by interested amateurs. “Perhaps the wheel is turning again where more people can participate

DNA Boosts OLED Performance–Usher in “Bio-LEDs”?

2009-01-11T09:58:00.000-08:00

Salmon (yes the fish) DNA Boosts OLED Performance–Usher in “Bio-LEDs”?May 9th, 2006
recent report in the esteemed journal "Applied Physics Letters" (American Institute of Physics; http://journals.aip.org) said that researchers in the US incorporating a thin layer of salmon DNA into the structure of a conventional OLED makes it ten times more efficient and thirty times brighter than their conventional counterparts. (Applied Physics Letters 88 171109)
Steve SechristSenior Analyst and Editorof Projection Monthly &Microdisplay Report
The team’s idea involves using the DNA as an electron-blocking layer. This improves the probability of electrons and holes recombining and emitting photons, which in turn enhances the device’s luminance.
"It turns out that DNA has nearly ideal energy levels that allow hole transport to proceed unimpeded while it prevents electrons being transported too quickly,"
Andrew Steckl from the University of Cincinnati told optics.org. "This gives both electrons and holes a greater opportunity to recombine and emit photons."
The report went on to say, the team tested a green- and blue-emitting BioLED against conventional OLEDs and found that the DNA electron-blocking layer improved the luminance in both cases. For a current density of 200 mA/cm2, the green BioLED achieved 15,000 cd/m2, whereas the baseline device reached just 4,500 cd/m2. The blue BioLED had a luminance of 1,500 cd/m2 at 200 mA/cm2, while the corresponding baseline device reached around 800 cd/m2.
Steckl and colleagues used DNA from Japan. "Salmon fishing is a very large industry in Hokkaido, Japan, some 200K tons per year," explained Steckl. "While the meat and eggs are edible, the male roe [sic] is normally a waste product but it is very rich in DNA."DNP
We had to take a second look at this story before deciding to go with it. The fact that the report was written up in Applied Physics Letters, and features Dr. Steckl the 2006 recipient of the Rieveschl Award for Distinguished Scientific Research brings significant credibility.
While our call to Dr. Steckl’s office at U of C wasn’t returned in time to add details to the article, we do plan to follow-up in our monthly Mobile Display Report issue, post haste. In the mean time, Matt Brennesholtz, IM’s resident scientist (who claims no expertise in OLED physics) speculates that it’s the nature of the long chain molecule of DNA with its double helix structure that offers the properties desirable for an electron-blocking layer. He also added that this is not growing LEDs or even living material, so the moniker "Bio-LED" is probably not appropriate.
We’re also curious to learn just how the team discovered that DNA’s characteristics match those properties needed for electron blocking in OLED’s. But certainly one lesson not lost on the professor is that answers can come from the strangest places. If these results are correct, does that mean my next OLED cellphone display will have male salmon milt in it? Is it possible to impregnate a display? Can such an OLED display spawn? What would the little feller look like? We can keep going, but we better stop here before we get in trouble.

Routine DNA testing in fish industry to help people and fish

2009-01-11T09:57:00.000-08:00

Routine DNA testing in fish industry to help people and fish
Over 1000 fish species can be legally sold in the United States, a challenge for accurate labelling. Many fish products such as fillets cannot be identified to species, even by experts. DNA surveys suggest that at least for some expensive species, most fish products are mislabelled. In 2004 Nature 430:309, scientists at University of North Carolina analyzed mtDNA of fish labelled as red snapper, which by US law can only be applied to a Caribbean snapper species, Lutjanus campechanus. 77% (17/22) fish purchased from 9 vendors in eight states were not L. campechanus, and most were species from other regions of the world, or could not be identified to species due to lack of reference sequences.
More recently, the availability of commercial DNA testing has enabled enterprising news stations to do their own research. Last year a Florida television station found that 6 of 11 restaurant entrees labeled as local grouper were other species, including Asian catfish and tilapia, and last month a Los Angeles television station reported that red snapper entrees at 4 local restaurants were either tilapia, catfish, or mahi mahi. Following up on the news media, the Florida Attorney General’s office did their own testing, found 17 of 24 restaurants sold entrees mislabeled as grouper, and made legal settlements. What is needed is needed is a widely available method backed up by a reliable reference library that can be routinely applied to identification of fish and fish products in the marketplace. DNA barcoding is designed to be just that.
The Food and Drug Adminstration (FDA) Regulatory Fish Encyclopedia (RFE) aims “to assist with the accurate identification of species and help federal, state, and local officials and purchasers of seafood identify species substitution and economic deception in the marketplace.”
The species pages include scientific and common names, pictures of whole fish and fish products, analytic gels of fish proteins, and excitingly, an empty space for reference DNA sequence information. For reliable identification, the fish reference library needs comprehensive taxonomic coverage and adequate sampling of variation within species, ie DNA barcoding. I believe the Fish Barcode of Life Initiative (Fish-BoL), which has already collected barcodes from over 16,000 specimens representing more than 3500 species, will provide a widely used tool that will benefit consumers and the many species of fish that require management or protection.

DNA helps sort out really big animals

2009-01-11T09:56:00.000-08:00

DNA helps sort out really big animals, crowding ArkDNA identifies invasive parasitic wasp »Freshwater fish DNA data debut
In June 2008 PLoS ONE, thirteen researchers from nine Canadian universities, museums, and federal agencies report on mtDNA sequences from 1360 individuals representing 195 (95%) of Canada’s 205 freshwater fish species. Hubert et al follow “best practices” established for DNA barcode records (similar criteria would enhance the value of other genetic reference data as well), namely each sequence is derived from a vouchered specimen and the barcode record includes:
* “Bi-directional sequences of at least 500 base-pairs from the approved barcode region of COI, containing no ambiguous sites * Links to electropherogram trace files available in the NCBI Trace Archive * Sequences for the forward and reverse PCR amplification * Species names that refer to documented names in a taxonomic publication or other documentation of the species concept used * Links to voucher specimens using the approved format of institutional acronym:collection code:catalog ID number”
The researchers analyzed an average of 7.6 specimens/species, with an effort to sample across species ranges. A first pass look at genetic distances among and within Canadian freshwater fish shows results similar to those of other animal groups: average variation within species, 0.3%; average minimum distance between congeneric species (nearest neighbor), 8.3%; species with overlapping mtDNA sequences, 7% (4 species pairs and 1 flock of 5 species; one of the overlapping species pairs represents probable introgression. ) Five species showed divergent clusters differing by 1-2% in different parts of their geographic ranges, and 2 species showed larger divergences (3%, 7%); some or all of these might represent distinct species.
A challenge for science publishing is disseminating the large data sets that are increasingly generated. Restricting publication to only those studies with novel findings can lead to a kind of distortion, sometimes with serious consequences. The bias against negative studies, for example, is one factor contributing to the misculation of risks of medicines. As biodiversity genetics moves forward, we need ways to ensure high-quality work, receive appropriate academic credit, and disseminate results in a timely manner. PLoS ONE describes itself as “an international, peer-reviewed, open-access, online publication…that welcomes reports on primary research from any scientific discipline.” It seems to me that this sort of forum with a focus on quality rather than novelty is needed as a home for publication of large genetic data sets including DNA barcode records. Making this information available in a timely manner will in turn help drive development of analytic and display tools and enable scientific applications, such as identification of fish eggs and larva shown above.
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DNA dentifying Canadian Freshwater Fish Through DNA Barcodes

2009-01-11T09:54:00.000-08:00

dentifying Canadian Freshwater Fish Through DNA Barcodes
ScienceDaily (June 19, 2008) — New research by Canadian scientists, led by Nicolas Hubert at the Université Laval in Québec brings some good news for those interested in the conservation of a number of highly-endangered species of Canadian fish.See also:Plants & Animals
* Fish * New Species * Marine Biology
Earth & Climate
* Ecology * Biodiversity * Exotic Species
Reference
* Fish migration * Deep sea fish * Crappie * Conservation status
The use of DNA for automated species-level identification of earth biodiversity has recently moved from being an unreachable dream to a potential reality in the very near future. The potential of mitochondrial DNA in achieving this target has been successfully assessed for all of the Canadian freshwater fish communities and the approach bears some very exciting promise.
The Consortium for the Barcode of Life (CBOL) and the Canadian Barcoding of Life network recently assessed the potential of the Barcode region in diagnosing the entire freshwater fish communities of Canada and Alaska in the context of the fish worldwide campaign.
Hubert and colleagues sampled and barcoded 1360 individuals from 190 species belonging to 27 families and 20 orders and showed that Barcodes are effective for species-level identifications in 93% of the case.
In front of the economic importance and identification challenges associated with fishes, this represents a considerable advance for conservation practices and open new perspectives in ecology.
__________________________________------Mass Extinction Of Freshwater Species In North America
ScienceDaily (Sep. 30, 1999) — The first estimate of extinction rates of North America's freshwater animals shows that they are the most endangered group in the continent. "A silent mass extinction is occurring in our lakes and rivers," says Anthony Ricciardi of the study in the October issue of Conservation Biology.See also:Plants & Animals
* Extinction * New Species * Endangered Animals
Trends & Issues
Reference
* Zebra mussel * Fish migration * Mussel * Atlantic salmon
This research was done by Ricciardi of Dalhousie University in Halifax and Joseph Rasmussen of McGill University in Montreal.
Ricciardi and Rasmussen found that common freshwater species from snails to fish to amphibians are dying out five times faster than terrestrial species. In fact, freshwater animals are dying out as fast as rainforest species, which are generally considered to be the most imperiled on Earth.
If nothing is done, Ricciardi and Rasmussen predict that nearly 4% of freshwater species will be lost each decade. At this rate, many at-risk species will disappear within the next century. Currently, at-risk species account for 49% of the 262 remaining mussel species, 33% of the 336 remaining crayfish species, 26% of the 243 remaining amphibian species, and 21% of the 1021 remaining fish species.
One major threat to freshwater animals is non-native species. For instance, zebra mussels introduced from Europe are outcompeting native mussels in lakes and rivers.
Another major threat to freshwater species is dams. In the contiguous U.S., only about 40 rivers longer than 125 miles remain free-flowing. The fact that hundreds of U.S. dams are coming up for federal re-licensing soon gives us the unprecedented opportunity to re-establish natural flows in many rivers, say the researchers.

DNA mitochondrial

2009-01-11T09:53:00.000-08:00

"Even though several million species of plants, animals and microbes have been identified over the past 300 years or so, we still find new species,” Collette says. “And despite advances in technology, we still have to sort organisms into piles and research every known bit of information before we can say we found a new species. Sometimes we have to go back to the basics and look at an organism in a jar for reference to make a determination.”
Scientists like Collette often go to sea and collect dozens of samples of an organism they think might be a new species. They return to the lab to sort out what they have collected before focusing in on the details to determine exactly what the organism is, sharing samples with colleagues to reach a consensus. The process takes time, and they are excited about the contributions FISH-BOL and other similar efforts around the world will make to documenting and understanding life forms on earth.
After decades of following similar taxonomic procedures often done by visual identification, DNA barcoding offers a new and much faster, more accurate way to identify species and share information. Since nearly all biological species have distinct gene sequences, they can be identified using a short gene sequence collected from a standardized position in the genome – a DNA barcode. Barcoding of animals relies on differences between species in a relatively short segment of mitochondrial DNA.
The first step in FISH-BOL and other similar efforts is to build a public library of barcode sequences from museum reference specimens. Specimens provide tissue samples that produce a reference barcode for that specific species. Researchers using standard tools and techniques in molecular biology extract DNA from the tissue, locate and isolate the barcode region, then replicate and sequence the genes. Then the specimens, known as voucher specimens, are preserved and catalogued into archival museum collections.
Not only can specimens be identified as part of a known species using only a tiny piece of tissue from the organism, but new variations in what was thought to be a single species can be determined. Tissue from unidentified specimens can also be matched to the DNA sequence of a known species in the reference library, a process that currently takes just a few hours and a few dollars but soon may take only minutes and cost pennies.
Barcode records of each specimen contain the DNA sequence, information about the voucher or referenced specimen, and the species name. The records are stored in three global databases and are available without charge. As an electronic database, FISH-BOL contains DNA barcodes, images and geographic information of examined specimens, as well as linkages to the voucher specimens, information on species distributions, nomenclature, taxonomic information, natural history information and literature citations.
"Barcoding works for all stages in the life cycle, so it will help us identify larval fish, which is timely since many leaders in larval fish taxonomy are retiring,” Collette said. “It can also differentiate between closely related species that are hard to tell apart, especially large specimens that are difficult to bring back from the field. And it can positively identify fishery products like fish fillets so you know if the grouper you ordered in a restaurant is really grouper."

Building A Global Reference Library Of DNA Barcodes Of Marine Life

2009-01-11T09:51:00.000-08:00

Building A Global Reference Library Of DNA Barcodes Of Marine Life
ScienceDaily (Apr. 23, 2008) — Most of us are familiar with bar codes, those small black stripes with numbers below, known as the Universal Product Code or UPC label, that appear on commercial products. We scan them at the grocery store or to check a price, or have to cut them out and send them in for a rebate.See also:Plants & Animals
* New Species * Fish * Nature * Wild Animals * Endangered Animals * Extinction
Reference
* Fish migration * Fishery * Marine conservation * Deep sea fish
Now imagine scanning a DNA barcode on the piece of fish you just bought for dinner to instantly verify the species, where it came from, its nutritional value, and other valuable information. NOAA researchers are helping to make this scenario a reality.
"We need to accurately identify species for a number of reasons, from documenting the biodiversity of poorly sampled species and geographic areas to understanding populations and managing global fisheries in a sustainable way,” said Bruce Collette, a zoologist at NOAA’s National Systematics Laboratory (NSL) located in the Smithsonian Institution in Washington DC. "DNA barcoding is another tool in the toolbox of taxonomists and researchers who study, document, and organize knowledge about all life forms on earth.”
Collette and colleagues at NSL, part of NOAA’s Northeast Fisheries Science Center, are participating in FISH-BOL, the global Fish Barcode of Life Initiative, which plans to collect at least five representatives each of all 30,000 plus marine and freshwater species in the world. FISH-BOL is part of the global Consortium for the Barcode of Life (CBOL), started in 2003 to barcode everything from fishes, mushrooms and flowers, to microbes, insects and animals of every description.

DNA ISA DOUBLE STANDED MOLECULE

2009-01-11T09:48:00.000-08:00

DNA is a double stranded molecule, and when it is chemically denatured and separated into two strands, it quickly reanneals into a double stranded conformation. Thus, when a single stranded probe is incubated with a single-stranded (denatured) metaphase chromosome (or interphase), the probe will bind to complementary DNA sequences to reform the double stranded molecule. Overall, the most critical step when using FISH is the choice of adequate probes. A DNA probe is defined according to its target or complementary DNA in metaphase and interphase cells: (1) repetitive sequence probe, (2) whole chromosome (painting), (3) locus-specific probes.
Technically the ideal probes especially for interphase FISH should give strong, specific signals with no backgrounds and should have a high hybridization efficiency (> 90%).MaterialsReagents
For slides:0.005%Pepsin/0.001M HClPost fixation wash (5 ml PBS 10X, 5 ml MgCl2 0.5M, 40 ml H2O)Paraformaldehyde/PBS (5ml PBS 10x, 5 ml MgCl2 0.5M, 15 ml H2O, 25 ml Paraf. 8%)
For probes:Buffer 10X (for 10ml: 5mL Tris-HCl 1M, 1mL MgCl2 0.5M, 0.005 gr BSA, 4 mL H2O)Cot-1 DNA (Invitrogen, 15279-011),Salmon sperm DNAHybridization Mix:For 15 ml:7.5ml pure formamyde, 6.0 ml Dextran Solfate 25%,1.5 ml 20X SSC. Keep it refrigerated.
LABELLING CY-3 (Amersham, FLUOROLINK CY3-dUTP, code: PA53022-25005442)LABELLING FluorX (Amersham FluorXdCTP code 11-0026-10)SSC solution
DAPI( ROCHE, 236 276)Antifade DABCO (SIGMA, D2522)Equipment
slidescoplin jarSavant centrifugeHYBrite (Vysis)LEICA DMRXA fuorescent mycroscopeTime Taken
Procedure
SLIDES PREPARATION1)Clean slides with 70% ethanol (to remove any grease or dust).2)Place a few drops of pellet and let the slide air dry.3) Place the slide in an incubator at 90°C for one hour and a half to age, or at 37°C overnight. In case they need to be used much later, place them at –20°C for indeterminate time.
SLIDES TREATMENT1)After 1.5 hours in the incubator, let the slide cool and then add 150-200 µl of 0.005%Pepsin/0.001M HCl and place them at 37°C for 15 min.2)Place them in a coplin jar with PBS 1x for 5 min.3) 5 min in post fixation wash4) 5 min in Paraformaldehyde/PBS wash5) 5 min in PBS 1x6) 5 min each in ethanol series: 70%,90%, 100% (slides can be left in 100% ethanol until ready to be hybridized).7) Before hybridization with probe, slides are taken out and let air dry completely.
LABELLING OF PROBEDIRECT LABELLING (NICK TRANSLATION)FOR 1? OF DNA
10ul DNA3ul Buffer 10X0.6ul dAGC (for dUTP/CY-3, red) or 1.8ul dAGT (for FluorX-dCTP, green)0.3ul dUTP/CY-3 or 0.9ul for FluorX-dCTP3ul B-mercaptoethanol0.3ul DNApolymerase6ul DNAse (1:700 ul H2O)H2O to reach 30ul final volume
1)After preparing the labelling mixtures with enzymes and DNA, place it in a water bath at 16°C for 2 hours.2) After 2 hours, to PRECIPITATE THE PROBE take the appropriate quantity of labelled probe and place it in a new eppendorf (for BAC probes 30 ul)3)Add to the labelled probe:3 µl Salmon Sperm DNA (SSD)10 µl Cot-1 DNA (10µl of Cot per 30µl of labelled DNA, so for a cosmid add 15 µl)1/10 Vol NaAC3 Vol cold EtOH 100%
PRECIPITATION1)To precipitate, place eppendorf at –80°C for 15 min, or at –20°C for at least 30 min.2) Centrifuge at +4°C for 20 min.3)Take supernatant off and dry the pellet (Savant centrifuge could be used to dry pellet in a better way)4) Resuspend pellet in Hybridization Mix5) Place in thermomixer at room temperature for 10 minutes to mix.6) Place probes on dried slides7)Place coverslip and seal it with rubber cement.8)Place slides in Hybrite (Vysis) and start cycleHybrite temperatures: Melt: 69°C for 2 min(human slide) Hyb: 37°C Overnight
CO- HYBRIDIZATIONTwo probes are labelled with different fluorochromes and hybridized on the same slide: CY3-dUTP(red) and FluorX-dCTP (green). They can be easily used because they are both direct labelled probes.Each probe is labelled individually, and then they are mixed together and precipitated.Example: 30 ul labelled BAC A plus 30 ul labelled BAC B plus 3 ul SSD plus (10ul plus 10ul) 20 ul Cot-1 DNA plus 8.3 ul NaAc plus 270 ul EtOH.The rest is the same as the single hybridization protocol.
RE-HYBRIDIZATIONA slide can be re-hybridized up to four times (at least) with good results.1) Remove coverslip from slide2)Place slide for 2 hours at 42°C in 2x SSC3) Rinse in PBS 1x at room temperature.4)Place slide for 5 min in each Ethanol: 70%, 90%, 100%5)Decrease denaturation time.Hybrite Temperatures: Melt: 69°C for 1 min(human slide) Hyb: 37°C Overnight
POST-HYBRIDIZATION WASHES
• 3 washes at 57°C in 0.1x SSC for 5min each• 5 min in DAPI (60 ml of 2x SSC, 120 ul DAPI)• Add a few drops of Antifade DABCO on the coverslip and make sure there are no air bubbles between slide and coverslip
ANALYSISAnalyse with LEICA DMRXA Fluorescent microscope.Images can be acquired using applied spectral imaging (ASI) camera and analyzed with FISH view 2.0 softwareTroubleshooting
1)Before probe resuspension in the Hybridization Mix it is important to pre-resuspend the precipitated probe in a few ul of H2O.2)Pepsin treatment is very important for chromosome quality: the amount of pepsin used and the length of pepsin treatment need to be very precise.3) It is better to add cold paraformaldehyde in the paraformaldehyde/wash.Critical Steps
Anticipated Results
References
1) Melixetian et al. Loss of Geminin induces rereplication in the presence of functional p53. J.Cell.Biol 2004 May 24; 165(4):473-822) Minucci et al. PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors. _Blood- 2002 Oct 15; 100(8):2989-95__________________________________________

DNA barcoding

2009-01-11T09:46:00.000-08:00

Universal primer cocktails for fish DNA barcodingNATALIA V. IVANOVA*, TYLER S. ZEMLAK, ROBERT H. HANNER and PAUL D. N. HEBERTCanadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada N1G 2W1Correspondence: N.V. Ivanova, Fax: (519) 824 5703; E-mail: nivanova@uoguelph.caCopyright © 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing LtdKEYWORDSCOI • degenerate primers • M13-tailed primers • species identificationABSTRACT
Reliable recovery of the 5' region of the cytochrome c oxidase 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all fish species. In this study, we develop and test primer cocktails with a view towards increasing the efficiency of barcode recovery. Specifically, we evaluate the success of polymerase chain reaction amplification and the quality of resultant sequences using three primer cocktails on DNA extracts from representatives of 94 fish families. Our results show that M13-tailed primer cocktails are more effective than conventional degenerate primers, allowing barcode work on taxonomically diverse samples to be carried out in a high-throughput fashion.