Ocean ‘Dandruff’ DNA to Better Study Marine Biology

  • DNA Barcoding for all Organisms has Numerous Applications
  • DNA Barcodes from Water Samples Greatly Aide Marine Biologists
  • Aquatic Environmental DNA (eDNA) Proves to be Informative ‘Dandruff’

Human DNA identity analysis is now commonplace methodology that’s frequently featured in newspaper stories, TV crime series, or “who dun it” movies. The same principle (i.e. using a characteristic DNA pattern or signature) applies to identification of all animals, birds, insects, and microbes. Actually, DNA barcoding extends to any organism, whether it is alive or has been dead for hundreds of thousands of years (so long as it’s preserved by fossilization).

Taken from gajitz.com

Marine biologists face a serious challenge with accounting for very diverse forms of marine life that exists in a mindboggling huge volume of water. Consequently, it’s not surprising that analysis of water-borne, marine DNA barcodes—as proxies for going to and counting fish—is rapidly trending in utility and importance. Known formally as environmental DNA (eDNA), the aquatic version has been humorously referred to as ocean ‘dandruff’ by Christopher Jerde of the University of Nevada in Reno (which, ironically, is landlocked and distant from any ocean.) But I digress. Before diving further (pun intended) into ocean dandruff, let’s briefly review the background of DNA barcoding.

DNA Barcodes 101

Prof. Paul Herbert. Taken from uoguelph.ca

In 2003, Prof. Paul Herbert and coworkers in the Department of Zoology at the University of Guelph in Canada published a seminal study titled Barcoding animal life: cytochrome c oxidase subunit 1 (CO1) divergences among closely related species that fundamentally changed the field of taxonomy. In a nutshell, Herbert’s team showed it was feasible to classify millions of species based only on DNA sequence of the mitochondrial gene CO1. In the intervening, relatively short amount of time, there have been thousands of publications dealing with applications and extensions of this concept, which is now recognized to be very powerful and promising albeit with some limitations.

Typically, DNA barcodes are identified by sequencing after PCR amplification of one or more specific genetic loci such as CO1. Following proof that a DNA barcode can differentiate the species of interest, single- or multiplex quantitative PCR (qPCR) can be used to enumerate relative amounts of sample from the field.

The advent of high-throughput sequencing technologies applicable to complex mixtures of individually tagged samples then gave rise to “metabarcoding,” about which interested readers can consult many publications for specific topics.

Craig Venter steers his research yacht, Sorcerer II, under the Sydney Harbour Bridge in his quest to collect microbes from the world’s waters. Photo: Dallas Kilponen. Taken from smh.com.au

BTW, among the many pioneering scientific ventures by uber-famous Craig Venter, is his Global Ocean Sampling Expedition aboard his research yacht, Sorcerer II. The expedition is a quest to unlock the secrets of the oceans by sampling, sequencing and metabarcoding DNA of all (or most) microorganisms living in these waters.

Lest you think this was a well-intended but unproductive journey—some say junket—by Venter and coworkers, here’s a link to peruse 16 resultant publications that I found by searching PubMed. To watch and listen to Venter talk about this work, you can click here for an educational and entertaining—as usual with Venter—TED Talk on Sampling the Ocean’s DNA that’s had over 550,000 views!

Ocean ‘Dandruff’

Now that we’ve covered the basics of DNA barcoding and metabarcoding, let’s turn back to ocean dandruff. Dandruff, simply put, is dead skin cells. Using dandruff as an intended witty metaphor for ocean eDNA is a bit misleading as marine eDNA is comprised of a complex mixture of cellular matter from scales, feces, decomposing tissue, etc. of fish and all other present or past sea creatures. Consequently, the design and specificity of primers for PCR is of paramount importance for obtaining—let alone interpreting—DNA barcodes based on fragment size or sequence.

As reported by Miya et al., monitoring the occurrence of fish species-specific eDNA PCR fragments (~70–300 bp) has traditionally used conventional electrophoretic gel separation and detection. More recently, qPCR using fluorogenic probes has been employed owing to the method’s sensitivity, specificity and potential to quantify the target DNA. For example, it has been possible to accurately estimate the biomass of common carp in a natural freshwater lagoon using qPCR of eDNA concentrations and biomass in aquaria and experimental ponds.

Miya et al. also describe the development of a set of PCR primers for metabarcoding mitochondrial DNA of 880 species of fish. They sampled eDNA from four tanks with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing. Out of the 180 marine fish species contained in the four tanks, they detected 168 species (93.3%) distributed across 59 families and 123 genera. That’s quite an impressive accomplishment.

Ocean Dandruff Case Studies

Since there are so many fish-related applications of DNA barcodes, I’ve selected several recent examples that are indicative of the utility of ocean ‘dandruff’—and are quite interesting, in my opinion. The first case in point exemplifies how eDNA can be used to deal with rare and endangered species, which are either very hard to find or can be dangerously distressed by catching to obtain samples.

Green SturgeonBergman et al. report that a decline in abundance of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning by these Green Sturgeon are effective at monitoring fish densities in concentrated pool habitats, results do not scale well—pun intended. By contrast, eDNA provides a relatively quick, inexpensive tool to efficiently identify and monitor Green Sturgeon DNA.

Taken from mthsecology.wikispaces.com

These investigators concluded that follow-on work based on this first-ever eDNA study of Green Sturgeon has the potential to provide better knowledge of the spatial extent of Green Sturgeon spawning that could help identify previously unknown spawning habitats and discover factors influencing habitat usage, guiding future conservation efforts.

Monterey Bay—The second case study, by Port et al., involves taking stock of the marine mammals and fish in Monterey Bay using eDNA and, importantly, comparing the results obtained to those from traditional dive surveys.

In brief, this team of researchers from several universities and the Monterey Bay Aquarium Research Institute found that eDNA assessments picked up almost all the organisms scuba divers spied underwater—plus many more that human eyes missed. Here’s some detail on how they did this.

At each scuba survey location as well as at sites offshore, ~1 gallon of water was sampled several feet above the bottom. Four types of habitats were sampled: sea grass beds, Monterey Bay’s unique “Kelp Forest,” sandy areas and rocky reefs. Onshore, in a “clean” (DNA-free) lab, these water samples were filtered to collect cells containing eDNA for storage at −80 °C until eDNA extraction at a university clean lab. A vertebrate‐specific primer set targeting a small region of the mitochondrial DNA 12S rRNA gene was used for PCR followed by gel purification.

Researchers collecting water in Monterey Bay for eDNA analysis. Courtesy Jesse Port. Taken from mercurynews.com

After quantification, pooled amplicons (each having a sample index sequence) were paired-end sequenced on the Illumina MiSeq platform using a 20% PhiX spike‐in control to improve the quality of low‐diversity samples. The conclusions are worth quoting because—in my opinion—the findings represent a new era in marine biology based on nucleic acid analysis:

“We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 meters. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.”

Restated more simply, eDNA analysis of the water picked up 11 of the 12 fish and marine mammals that the divers observed, and—importantly—identified 18 additional animals the divers missed! The efficiency and improvement offered by eDNA analysis compared to traditional seek-and-count methods has been echoed in an editorial I found by Hoffmann et al. titled, tongue-in-cheek, Aquatic biodiversity assessment for the lazy.

Invasive Gobies—The third and final case study deals with detection of invasive, non-native fish to assess whether eDNA can provide a better advanced warning system for detecting these unwanted creatures and implementing eradication steps.

Gobies are an invasive fish species that has colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby (Neogobius melanostomus), pictured below, is among the worst invaders in Europe. Current methods to detect the presence of these gobies are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile.

Taken from animal.memozee.com

To improve monitoring, Swiss and Canadian collaborators developed an assay based on the detection of eDNA in river water, without detecting any native fish species, which is obviously an important assay criterion. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by novices and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s reports and fish community monitoring.

I could go on and on with examples of utility and the many advantages provided by eDNA for marine biology, but I’m sure you get the picture. I hope that you agree with me that eDNA analysis is a very valuable type of trending nucleic acid-based methodology.

As usual, your thoughts or comments are welcomed.





DNA Day 2017

  • There are Now Millions of DNA-Related Publications
  • Some of the Top 5 Cited Papers on DNA Will Surprise You
  • You Probably Won’t Guess Top 5 Most Frequently Cited

Deciding what to post here in recognition of DNA Day 2017 was just as challenging as it has been in past years, primarily because there’s so many different perspectives from which to choose. After much mulling, and several abandoned approaches, I settled on featuring DNA publications that have received the most citations, as an objective metric—not just my subjective opinions about topics I think are significant or otherwise interesting.

Before getting to the numbers of DNA-related papers and some of the most cited papers, here’s a quick recap of what was posted here in the past, starting with the inaugural blog four years ago:

2013—60th Anniversary of the Discovery of DNA’s Double Helix Structure

2014—My Top 3 “Likes” for DNA Day

2015—Celebrating Click Chemistry in Honor of DNA Day

2016—DNA Dreams Do Come True!

Explosive Growth of DNA Publications

Regular readers of my blogs will know that I frequently use the NIH PubMed database of scientific articles to find publications by searching keywords, phrases, or authors. A convenient feature of these searches is providing “results per year” that can be exported into Excel for various purposes. Some preliminary searches indicated that DNA-related articles can be indexed by either DNA or PCR, or cloning, or other terms among which sequencing was notable. The majority, however, were indexed as either DNA or PCR, which together gave nearly 1.7 million items—an astounding number. This number is even much greater since PubMed excludes some important chemistry journals, as well as patents.

Diving deeper into these numbers, I thought it helpful to look at the publication volumes and rates for DNA, sequencing DNA, and PCR through 2015 starting from 1953, 1977, and 1986, respectively. These respective dates correspond to seminar publications by Watson & Crick, Maxam & Gilbert, and Mullis & coworkers. The results shown in the following graph attest to my often stated “power of PCR” as premier method in nucleic acid research, which we’ll see again below in another numerical context.

Top 5 Cited Papers

During my perusal of the above literature in PubMed generally related to DNA, I thought it would be interesting to find, and share here, which specific papers have the distinction of being most frequently cited. Citations are not available in PubMed, but are compiled in Google Scholar, which led me to these Top 5 that are listed from first to fifth.

Frederick Sanger (1918-2013) Taken from newscientist.com

  1. DNA sequencing with chain-terminating inhibitors

Frederick Sanger, the eponymous father of the “Sanger sequencing” method published in 1977, received the 1980 Nobel Prize in chemistry for this contribution. He also received the 1958 Nobel Prize in chemistry for sequencing insulin, and is the only person to win two Nobel Prizes in chemistry. Uber-famous DNA expert Craig Venter is quoted as saying that ‘Fred Sanger was one of the most important scientists of the 20th century,’ [who] ‘twice changed the direction of the scientific world.’

  1. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method

Kenneth J. Livak, PhD
Taken from archive.sciencewatch.com

The most commonly used method to analyze data from real-time, quantitative PCR (RT-qPCR) experiments is relative quantification, which relates the PCR signal of the transcript of interest to that of a control sample such as an

untreated control. The derivation, assumptions, and applications of this method were published in 2001 by Livak & Schmittgen. I overlapped with Ken Livak at Applied Biosystems, which pioneered commercilaization of RT-qPCR reagents and instrumentation at the time. He is currently Senior Scientific Fellow at Fluidigm Corp.

Sir Edwin M. Southern Taken from ogt.co.uk

3. Detection of specific sequences among DNA fragments separated by gel electrophoresis

Sir Edwin Mellor Southern, FRS, the eponymous father of “Southern blotting” DNA fragments from agarose gels to cellulose nitrate filters published in 1975, is a Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College. He is also Founder and Chief Scientific Advisor of Oxford Gene Technology.

  1. Prof. Bert Vogelstein, MD
    Taken from hhmi.org

    A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

This paper by Feinberg & Vogelstein published in 1983 describes how to conveniently radiolabel DNA restriction endonuclease fragments to high specific activity using the large fragment of DNA polymerase I and random oligonucleotides as primers. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments. His group pioneered the idea that somatic mutations represent uniquely specific biomarkers for cancer patients, leading to the first FDA-approved DNA mutation-based screening tests, and now “liquid biopsies” that evaluate blood samples to obtain information about underlying tumors and their responses to therapy (an area that I’ve touted in previous blogs). A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 109 dpm/μg of DNA can be obtained using relatively small amounts of precursor. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments.

  1. Kary B. Mullis, PHD
    Taken from TED.com

    Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

In 1988, Kary B. Mullis and coworkers (then at Cetus Corp.) published in venerable Science a method using oligonucleotide primers and thermostable DNA polymerase from Thermus aquaticus to amplify genomic DNA segments up to 2000 base pairs to detect a target DNA molecule present only once in a sample of 105 cells. Since that time, polymerase chain reaction (PCR)-related technology has evolved to now routinely enable a variety of single-cell analyses of DNA or RNA. Dr. Mullis received the 1993 Nobel Prize in chemistry for his 1983 invention of PCR, which his website says ‘is hailed as one of the monumental scientific techniques of the twentieth century.’

Top 5 Papers by Citation Frequency

While writing the above section, it occurred to me that ranking these five publications by total number of citations-to-date in Google Scholar doesn’t account for differences in the number of years between the year of publication and now. I did the math to calculate the average citation frequency per year, and here’s the totally surprising—to me—result: relative gene expression methodology published by Livak & Schmittgen is by far the most frequently cited of the Top 5, according to this way of ranking:

  1. 2001, relative gene expression, Cited by 69560 = 4,637 avg. citations per year
  2. 1977, Sanger sequencing, Cited by 32662 = 1,701
  3. 1975, Southern blotting, Cited by 21201 = 796
  4. 1988, PCR, Cited by 18785 = 671
  5. 1983, oligolabeled DNA, Cited by 21200 = 642

I should point out that, as transformative methods such as these gradually become widely recognized as “standard procedures,” researchers tend to feel it unnecessary to include a reference to the orignal publication. Consequenly, citation frequency decreases with time even though cummulative usage increases. In other words, 25 years from now average citations per year for relative gene expression will have likely decreased, and be surpassed by a new “method of the decade,” so to speak.

Prediction for the Future

This line of reasoning leads me to close with some speculation about what DNA-related technique might emerge as the next “method of the decade” that tops the above ranking by citation frequency.

My guess is that it will be Multiplex genome engineering using CRISPR/Cas systems by Zhang & coworkers that has been cited by 4145 at the time I’m writing this piece, only four years from its publication in venerable Science in 2013. Some of my blogs have already commented on various aspects of CRISPR/Cas9, which is among genome editing tools offered by TriLink.

As usual, your comments are welcomed.

Finding Frankfurter Fraud Featuring Famously Familiar PCR

  • While Thousands of PCR-based Tests for Food Authentication Exist, Commercial Adoption is Lax
  • PCR Tests for Halal Frankfurter Products Reported by Malaysian Team
  • PCR-enabled Next Generation Sequencing of USA Frankfurters Exposes Extensive Mislabeling and Adulteration

Regular readers of this blog will know that (a) I favor alliterations, (b) frequently feature PCR-based topics, and (c) I am fond of food facts involving nucleic acids, all of which are found in the title of this posting. While writing style and food are both a matter of taste, so to speak, it’s almost impossible to comment on nucleic acids without involving PCR, in one way or another, as PCR is—in my opinion—the most widely used and important method in molecular biology.

Having said this, and knowing that this summer alone Americans will likely consume an estimated 7 billion (!) hot dogs, (a.k.a. frankfurters or wieners), I thought it apropos to now feature finding frankfurter fraud by PCR. But before getting to that, I thought it’s worth commenting first on a frankfurter vs. wiener vs. hot dog and other “meaty” definitions to get us “linked” up—please pardon the puns, another of my penchants.

Frankfurter, Wiener, Hot Dog Lexicon

Frankfurter sausages and sauerkraut. Taken from tripadvisor.com

While it’s a fact that a resident of Frankfurt, Germany is properly called a Frankfurter, one of Frankfurt’s pork sausage specialties is also called a frankfurter—but spelled with lower case f—and is short for Frankfurter Würstchen, which go back to the 13th century. By the same token, wiener refers to a pork and beef sausage specialty introduced in the 18th century in Vienna, Austria—a city called Wien in German, and hence the word wiener.

Frankfurters and wieners look very similar, with the main culinary distinction being absence of serving with the bun, which by contrast is characteristic of a hot dog. Readers interested in the origin of the hot dog’s name and bun usage will learn at this link—pun intended—that there are various and widely different claims for this name and usage.

Hot dogs in buns. Taken from clowns4kids.com

Dog Factory, a short film by uber-famous Thomas Edison in 1904 poked fun at what went into hot dogs. Taken from wikipedia.org

In one such claim, the term dog is said to have been linked—there I go again—to sausages made from dog meat, as popularized in an old spoof by Thomas Edison (!) pictured below. This issue of what meat(s) hot dogs and similar sausages contain now segues into finding frankfurter fraud using PCR.

Finding Food Fraud by PCR

Before getting to the meat of this matter (oops!) involving frankfurters, I thought it would be informative to provide some larger perspective on finding food fraud, generally, utilizing the power of PCR for specific detection of nucleic acids that are characteristic of a given species of any food whether it be meat, fish, vegetable, etc. My search of Google Scholar for articles with all of the terms “food, fraud, and PCR” gave ~3,900 items. Here are some selected samplings, the first of which was taken from several I found that used TriLink products—hooray!

  • US Food & Drug Administration researchers employed species-specific primers from TriLink for multiplex real-time PCR analysis of salmon and trout species in a range of 80 commercial products in North America. 4 instances (5%) of fraud were found.
  • By contrast, a whopping 40% of commercial pet food products tested by PCR for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) were found to be potentially mislabeled, according to a study by academic researchers.
  • Saffron produced from dried stigma of Crocus sativus is considered to be the most expensive food spice in the world, as ~200,000 (!) flowers must be carefully hand-picked (!) to produce only 1 kg of spice. Combating saffron fraud with PCR has already led to ~150 publications (!).
  • PCR-based food authentication to screen for possible allergens or GMOs is important for prevention of potentially life-threatening food contamination or alleviating consumer perceptions—or perhaps misperceptions, as I’ve commented on previously.

Finding Frankfurter Fraud by PCR

As for finding frankfurter fraud by PCR, the aforementioned ~3,900 items from my Google Search of food, fraud, and PCR was sub-searched for frankfurter, which led to only 3 reports titled as follows:

The third item, which is by a team of Malaysian researchers and is the most recent, offers some interesting introductory perspectives on strict religious, cultural, or geographical restrictions over the consumption of certain meats in the context of commercial frankfurters.

For example, pork is totally unacceptable to Malaysia’s large Muslim population, as well as Jewish and certain Christian denominations. On the other hand, Egyptians prefer buffalo because of their cultural preferences, while some Indians and Europeans avoid beef because of religious requirements and the fear of bovine spongiform encephalopathy (aka “Mad Cow Disease”—click here for an FDA update), respectively.

In this Malaysian study, 100% beef, buffalo, or pork frankfurters were prepared as models, and then fully heat-processed in the laboratory to simulate conventional manufacturing procedures. Additional beef, buffalo, or pork frankfurter models were deliberately contaminated by “spiking” in 1%, 0.5%, or 0.1% of buffalo and pork, beef and pork, and beef and buffalo meat, respectively. PCR was then performed using species-specific primer pairs for two genes (cytochrome b and NADH dehydrogenase subunit 5) for cross-validation. Twenty different halal-branded (i.e. pork-free) “beef” frankfurters from Malaysian markets were tested. While no pork was detected in any of the tested “beef” frankfurters, they were all beef- and buffalo-positive, thus revealing that all of the investigated Malaysian commercial “beef” products were buffalo-adulterated.

Halal Certification by PCR

The above mentioned concern for non-pork halal-assurance piqued my interest as to the extent of PCR usage for halal-related certification, and my subsequent Google Scholar search for “halal, certification, PCR” gave nearly 350 items. This relatively large number of reports signals quite widespread adoption of PCR. I encourage those interested to peruse these items later, but will mention here that the following article is the most cited—close to 100 citations as of February 2017—Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

In closing, I think it’s worth noting in the context of halal certification—and increasingly popular “democratization” of technology, about which I’ve previously offered comments—a French start-up company now offers an antibody-based “dip stick” kit (Halal Test) for anyone to use for pork-free halal-assurance at home or even when eating out. Amazing.

Taken with copy write permission from French duo launch HalalTest: ‘We want to democratize analysis’ by Rachel Arthur+Rachel ARTHUR, 05-Nov-2014

Hot Dog! — There’s Now an NGS Food Authentication Service Company

Frankly—pun-to-be intended—I don’t know how “hot dog!” became an exclamatory phrase for good news. It applies, however, to the fact that Clear Labs Inc. (a 2014 start-up in Palo Alto, California) has proven that several issues of concern for consumers of hot dogs can be successfully addressed by PCR/NGS methods.

In a Clear Labs’ poster abstract for the 2016 International Association of Food Protection meeting, results were reported for a study of 345 hot dog products sold by national brands to compare product label information and ingredient lists with the results of NGS analyses. Following DNA extraction, universally accepted regions for animals, plants, and bacteria were PCR-amplified for NGS, which revealed that ~15% of these products had ingredient substitution, unexpected ingredients, or hygienic issues. In addition, 10% of all products labelled as vegetarian contained detectable levels of meat DNA. Vegetarian products also accounted for 67% of hygienic issues, such as human DNA.

At the risk of overly generalizing these findings, I believe that they probably reflect very widespread issues in the food industry, which we as consumers are virtually helpless to deal with, and can only hope that US FDA regulations begin to mandate PCR-based food certification.

Having said this, I didn’t want to end with a “downer.” Therefore, I’ll conclude with some hopefully fun facts about hot dogs, which—if you’re wondering—can be made from beef, pork, turkey, chicken, or a combination (but must be labeled as such, according to FDA information worth reading later).

Fun Frankfurter (aka Hot Dog) Facts

Just for fun, I calculated that the 7 billion frankfurters to-be-consumed by Americans this summer would stretch 56,818 miles (assuming each one were 6 inches long), which would wrap around Earth’s equator 2.3 times if placed end-to-end!

Some other fancinating frankfurter feats:

  • Taken from wikipedia.org

    The world’s longest hot dog was 197 feet and was prepared by Shizuoka Meat Producers and the All-Japan Bread Association for the latter’s 50th anniversary celebration in 2006 at the Akasaka Prince Hotel:

  • The world’s most expensive hot dog is the “California Capitol City Dawg”, served at Capitol Dawg in Sacramento, California and cost $145.49. Proceeds from the sale of each 18-inch long, 3-pound super “Dawg” are donated to the Shriners Hospitals for Children.
  • The annual Nathan’s Hot Dog Eating Contest is held on Independence Day at Nathan’s Famous original, and best-known restaurant in Coney Island, a neighborhood of Brooklyn, New York City. The current champion is Joey “Jaws” Chestnut, who ate a stomach-busting 70 (!) hot dogs and buns in only 10 minutes (!) at the 2016 championship.

Taken from elitedaily.com

As usual, your comments here are welcomed.

Evolving Polymerases to Do the Impossible

  • Polymerases Aren’t What They Used to Be! 
  • Scripps Team Evolves Polymerases That Read and Write With 2’-O-Methyl Ribonucleotides
  • Key Reagents for Romesberg’s “Molecular Moonshots” Are Supplied by TriLink BioTechnologies

Long-time devotees of these posts will likely remember a blog several years ago about Prof. Floyd Romesberg at the Department of Chemistry, The Scripps Research Institute who achieved a seemingly impossible feat. Namely, designing a new pair of complementary bases such that DNA replicating in E. coli would be comprised of six bases, thereby creating a six-base genetic code that is expanded from Nature’s four-base code.

Floyd E. Romesberg. Taken from utsandiego.com

More recently, Romesberg has cleverly outfoxed Nature once again, this time by evolving nucleic acid polymerases into mutant polymerases that can do what heretofore seemed impossible. He and his research team’s publication (Chen et al.) is a tour de force of experimental methodology that is not easily read, and is even harder to simply summarize in a short space like this blog. Consequently, I’ll first tell you what was accomplished, then give a short synopsis of principal new methodology, and close by commenting on the significance of this fascinating work.

Doing the Impossible

Romesberg’s lab successfully achieved what I think of as “multiple molecular moonshots,” wherein a Taq polymerase (which normally reads and writes DNA during PCR), was evolved by novel selection (SELEX) methods into mutant polymerases that are able to transcribe DNA into 2’-O-methyl (2’-OMe) RNA, and reverse transcribe 2’-OMe RNA into DNA for PCR/sequencing.

As depicted below, this was exemplified using a 60-mer DNA template and 18-mer 2’-OMe RNA primer to produce a fully-modified 48-mer 2’-OMe RNA by means of an evolved mPol and all four A, G, C and U 2’-OMe NTPs, which I’m proud to say were bought from TriLink BioTechnologies! This type of molecular evolution of a polymerase has no precedent.

DNA template   5’ ————————————- 3’

RNA primer                                 ←←← 3’ xxxxxx 5’

mPol ↓ 2’-OMe NTPs

Determining the fidelity of this seemingly impossible molecular transformation was addressed by achieving a feat of comparable impossibility! As depicted below, the aforementioned 48-mer 2’-OMe RNA product was hybridized to a DNA primer for reverse transcription into a 48-mer complementary DNA (cDNA) strand, using an evolved mPol, together with all four A, G, C and T unmodified dNTPS, which were also purchased from TriLink. This unprecedented conversion of 2’-OMe RNA into cDNA was followed by conventional PCR/sequencing, the results of which demonstrated relatively high fidelity.

2’-OMe template   5’ xxxxxxxxxxxxxxxxxxxxxxxxxxx 3’

DNA primer                                           ←←← 3’ —— 5’

mPol ↓ dNTPs

cDNA                        3’ ————————————– 5’

How They Did It

In the selection cycle shown below, (1) phage-display libraries were used to expose individual polymerases (Pol) on E. coli. cells in proximity to chemically attached primer/template complexes of interest, which are mixed with natural or modified triphosphates including biotin (green; B)-labelled UTP to extend the primer. (2) Phage that display active mutant polymerases (mPols) are isolated with streptavidin (SA) beads. After washing to remove nonspecific binders, phage cleaved from the beads are used to re-infect E. coli. (3) Heat-treated lysates of E. coli that express the recovered mPols are next subjected to plate-based screening using 96-well plates coated with primer/template complex and extension buffer that contained natural or modified triphosphates and B-UTP, incorporation of which is chromogenically detected. (4) Mutants that give rise to the most activity are selected for individual gel-based analysis, from which (5) promising candidates are selected for further diversification (e.g., by gene shuffling, as depicted) and then subjected to additional rounds of evolution.

Taken from Chen et al. Nature Chemistry (2017)

What is the Significance

In a previous blog, I’ve commented on increasing interest in the utility of aptamers, which are oligonucleotides that can specifically bind small molecules or motifs in proteins, and thus be used to build electronic sensors or studied as potential therapeutic agents rivaling antibodies. Therapeutic aptamers, like antisense oligonucleotides, require incorporation of chemical modifications to impart stability toward nucleases in blood or cellular targets.

Burmeister et al. have previously reported methods for mPol transcription of a DNA template into a fully modified, nuclease-resistant 23-mer 2’-OMe RNA aptamer—also using TriLink’s 2’-OMe NTPs! However, they encountered considerable experimental difficulties in generating this therapeutically promising 23-mer against vascular endothelial growth factor. These technical issues have now been surmounted by the mPol-evolution approaches in the present work by Romesberg’s team, which enabled improved access to longer 2’-OMe RNA aptamers with reasonable efficiency and fidelity.

Moreover, the present study is the first to evolve an mPol for reverse transcription of fully modified 2’-OMe RNA into DNA, which can then be amplified by PCR and/or sequenced, thereby opening the door for a variety of new analytical methods. Most importantly, the molecular mechanism by which these remarkable mPol activities was evolved, namely, the stabilization of an interaction between the “thumb and fingers domains,” may be general and thus useful for the optimization of other Pols. In that case, we can look forward to further advances in evolving other Pols to do the impossible—hopefully using modified nucleotide triphosphates from TriLink!

As usual, your comments are welcome.

Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from gene-quantification.de

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.


In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab


Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.


A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from tamu.edu

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Death of DNA Dogma?

  • Current Genetic Dogma is DNA → RNA → Protein
  • Two Research Teams Independently Implicate Sperm Short RNA Can Transmit Paternal Genetics
  • More Research Needed to Elaborate the New Dogma

The Central Dogma of all life on Earth is currently understood to be DNA encoding RNA that in turn encodes protein. That genetic inheritance is transferred as DNA was first posited by uber-famous Francis Crick, who coined the term Central Dogma. While dogmatic principles, by definition, should have no exceptions, a few species of viruses can be considered to be exceptional cases in this regard.

The Central Dogma. Taken from biology.tutorvista.com

The Central Dogma. Taken from biology.tutorvista.com

That said, there is now quite a scientific buzz—if not shudder by some—over reports implicating RNA molecules as direct (i.e. non-DNA) agents for mammalian inheritance. My instantaneous mental responses to these surprising—if not shocking—revelations was first, “Wow, who would have thunk?” and then, “I’ve got to share this news in a blog.” So here it is.

Surprising Science in Sperm

Human sperm. Taken from leavingbio.net

Human sperm. Taken from leavingbio.net

While most of us are probably at least passingly familiar with textbook descriptions of the basic structure of sperm and its functional role in reproductive molecular biology, more detailed information on its nucleic acid content is less known. Consequently, shown below is a depiction of the basic structural components of a sperm, DNA content, and primary functions for doing its job, so to speak, in fertilization of an egg.

By way of background, here’s information that I thought was worth sharing. My Google Scholar search results for nucleic acid content of sperm included a very impressive technological accomplishment reported by uber-famous professor/entrepreneur Stephen Quake and co-workers in 2012 on microfluidic separation methods for the first ever genome-wide single-cell DNA sequencing of human sperm. Contrary to what one might intuitively expect, 91 genomes of sperm from a single individual were not identical. Since DNA from only one sperm and one egg combine during fertilization, the exact paternal DNA genotypes in the resultant offspring involves “pot luck,” so to speak.

Regarding RNA, my Google Scholar search led to a paper in 2011 by Krawetz et al. on the first ever report of deep-sequencing of short (18-30 bases) RNA (sRNA) in human sperm (for which TriLink offers a high-performance CleanTag™ kit for sRNA library prep as detailed on this poster). Krawetz et al. found microRNA (miRNA) (≈7%), piwi-interacting RNA (piRNA) (≈17%), and repeat-associated sRNA (≈65%). A minor subset of sRNA within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. However, reproductive roles for this molecular menagerie (what I tongue-in-cheek call these various sRNAs) remain speculative.

Fast forwarding to present time leads us to the two “wow” publications in venerable Science that triggered this blog:

While you’ll need to read these publications for details, they collectively raise the following controversial question vis-à-vis the Central Dogma for strictly DNA-based inheritance.

Are You Inheriting More Than Genes from Your Father?

Yes, is the surprising—if not bombshell—answer to this question, which I borrowed from Mitch Leslie’s Science editorial headline. If this conclusion is supported by further studies, it forces a fundamental revision of reproductive molecular cell biology. That’s a very big deal, so to speak, with ramifications not to be under appreciated.

Using sRNA library preparation methods analogous to TriLink CleanTag™ for Illumina deep-sequencing, the USA-Canadian team analyzed sperm from male mice fed a low-protein diet, progeny of which showed elevated activity of genes involved in cholesterol and lipid metabolism. They found that >80% of sRNA were fragments from several kinds of transfer RNAs (tRNAs). Most notably, 5′ fragments of tRNA-Gly-CCC, -TCC, and -GCC shown below all exhibited an approximately 2- to 3-fold increase in low-protein sperm.

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

To understand when, where, and how these tRNA fragments were formed, as well as unravel functional significance, the researchers describe an experimental tour de force—in my opinion. This included antisense modified-oligonucleotide “knock-out” of these tRNA fragments, as well as “knock-in” injection of <40-nt sRNA populations purified from control and low-protein sperm into control zygotes.

The researchers concluded that the sperm acquired most of these fragments while passing through the epididymis, a duct from the testicle where the cells mature. Functionally, they also link tRNA fragments to regulation of endogenous retro-elements active in the preimplantation embryo.

In the second study, the China-USA team also found tRNA fragments by deep-sequencing of sRNA. After feeding male mice either a high-fat or low-fat diet, the scientists injected the animals’ sperm into unfertilized eggs, and then measured metabolic performance of the offspring, which ate a normal diet. Progeny of fat-eating fathers remained lean; however, they showed two abnormalities often found in their dads and in humans who are obese or diabetic—abnormal absorption of glucose and insensitivity to insulin.

Like the first study, these researchers also did “knock-in” experiments wherein they inserted the tRNA fragments into eggs fertilized with other sperm. Fragments that came from fathers that ate the high-fat diet resulted in offspring that also showed impaired glucose absorption.

Take Home Messages

At the risk of over simplifying or over generalizing, the aforementioned two studies of sRNA in sperm provide compelling—and stunning—evidence for how tRNA fragments in sperm are responsible for inheritance independent of sperm DNA sequences. So much for dogma.

With regard to specifics, researchers now need to investigate how permanent these changes are, and how quickly they can be reversed by changing diet.

The flip-side of a bad diet adversely influencing offspring is to investigate if and how a good diet imparts better health to offspring.

Please share your thoughts about these reports, conclusion, and implications by commenting here.


If you enjoy hip hop music—or just want to chuckle—this YouTube video for the Central Dogma song will get your head bobbing in sync with the music, lead you to smile, and give you a cool visual display of the central dogma.

National DNA Day 2016 – DNA Dreams Do Come True!

  • Khorana’s Dream of Synthesizing a Gene from Hand-Made Oligos
  • Caruther’s Dream of Automating Oligo Synthesis
  • Venter’s Dream of Fully Automating Gene Synthesis
  • Who’s Dreaming About What’s Next?

DNA Day ImageThis blog acknowledging National DNA Day on April 25th deals with dreams of various sorts, but mainly with gene synthesis, which was only a dream in the 1950s and is now achievable in a way few dreamed possible even a few years ago.

Before I get to DNA gene-dreams that did come true, I want to briefly mention two other dream-like anniversaries. First is the fact that my blog is now beginning its 4th year—yeh!—after its inaugural posting in April 2013 to celebrate 60 years since Watson & Crick’s famous publication of DNA’s helix structure as the fundamental basis for genetic material. Second is this year being TriLink’s 20th anniversary—yeh!—as a leading provider of modified nucleic acids, which co-founders Rick Hogrefe and Terry Beck likely view as their business dream come true. But I digress…

The First Dreamer and Doer Continue reading

Highlights of 2015 Publications Using TriLink BioTechnologies Products

  • Publications Citing TriLink Products Exceed 6,000
  • TriLink Products Showed up at a Rate of One Publication per Work Day
  • Among These Customer Publications, Modified mRNA is Trending
Taken from thetrymovement.com

Taken from thetrymovement.com

From my college classes decades ago, I can still clearly recall—thankfully—many “ah ha” moments. Most importantly is when I crystalized to purity and then confirmed structure by NMR the first compound I synthesized in Organic Chemistry Lab. Another ah ha moment—but on a completely different level—was during a philosophy class when the professor partially paraphrased a quote by Aristotle as “we are what we do.” The full quote given above is even more thought provoking because it ties in the notion of excellence, which I took to heart then, and have attempted to live by ever since.

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RNA in DNA—Mistake or Mystery?

  • Human DNA Misincorporates >1,000,000 Ribonucleotides Per Replication Cycle
  • These Mistakes are Likely Biological Mysteries
  • Four New Sequencing Methods May Demystify Why There’s “R in DNA”

When I came across a publication on the presence of RNA in DNA my initial reaction, frankly, was great surprise, if not outright disbelief. As the so-called “blueprint” of life, I reckoned that DNA is virtually sacred in terms of its chemical composition, albeit subject to base mutations as well as insertions and deletions of sequence. In other words, I had heretofore been under the impression that DNA’s repeating units are 100% deoxyribonucleotide (and conversely that RNA’s are ribonucleotides), thus giving DNA (and RNA) the eponymous name is has. So, I thought to myself, if that’s reportedly not the case for DNA, what are the facts and implications, i.e., is RNA in DNA just a rare “mistake” or is this yet another example of a “mystery” of Nature. Below is what I’ve learned about this revelation.
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We’re Celebrating Click Chemistry In Honor of National DNA Day

  • The Verbification of Click Chemistry 
  • Old Chemistry Morphs into New Applications for DNA and RNA  
  • Amazingly, Phosphorus in DNA and RNA is not Needed for Function 

This post comes only two days after National DNA Day 2015 on April 25th so it’s apropos to feature DNA, but I’d also like to give a nod to the lesser recognized RNA, without which DNA would be akin to music notes in search of a melody.  If you’re a regular reader of this blog, you know my stance on this subject and so I digress…

So-called “Click Chemistry” is trending so “hot” that it has led to a phenomenon known as verbification, which is when a noun becomes a verb by virtue of popularity and linguistic convenience. So, just as Google has become to google for virtually everyone, Click has become to click for synthetic chemists and biotechnologists. Whether or not you’re already familiar with Clicking, I hope to provide herein some interesting snippets about Click, its growing ubiquity, and how it has enabled synthesis of a completely novel, non-phosphorous linkage in DNA that nevertheless functions flawlessly in vivo—a stunning feat never before achieved that has intriguing implications about life. More on that later, but first some snippets about Click.

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