Tech News BioTechniques: Celebrating 30 Years of Methods Development Picture the world as it was in 1983, the year BioTechniques launched. Ronald Reagan was in the White House. Americans thrilled to the adventures of Luke Skywalker and Darth Vader in “Return of the Jedi” and took their first steps into the digital age with the then two-year-old IBM PC. On the scientific front, DNA sequencing was still relatively new. The polymerase chain reaction didn’t yet exist—it was still just a brainstorm inventor Kary Mullis had on a California highway that wouldn’t see print until 1985. Meanwhile, cell biologists were capturing ever more beautiful images with their microscopes, but their efforts were constrained by the then insurmountable diffraction limit. Not surprisingly, life science today bears little resemblance to the way it was practiced three decades ago. Whole-genome sequencing is now routine, as is PCR. And microscopists have breached the once inviolable diffraction limit, taking pictures of cellular structures with unprecedented resolution. To gain some perspective on the past 30 years in life science technology development, we spoke with key innovators in the fields of DNA sequencing, PCR, and microscopy. Their insights are both interesting and informative, a history lesson from the developers who changed the way we work in the lab.
Sequencing: Moving Up By Scaling Down In 1983 George Church was nearing the end of his graduate work at Harvard University. He had matriculated in 1977, and that same year joined the lab of DNA sequencing pioneer (and future Nobel laureate) Walter Gilbert. From the beginning, Church was focused on genome sequencing. In 1974, while still a Duke University undergraduate, he manually keyed in and folded the structure of every then-known tRNA sequence. “I started hoping that every person could afford to see their own DNA sequence,” he writes on his web site. Graduating in 1984, his thesis described “the first methods for direct genome sequencing, molecular multiplexing [and] barcoding.” That year, Church participated in a meeting at Alta, Utah that would launch the Human Genome Project (HGP). At the time, the only technology capable of decoding the human genome was Sanger dideoxy sequencing, and as the HGP took shape, some argued that the way forward was to ramp up existing technology. But Church argued for a different solution. “I felt that $3 billion was an unacceptable amount of money for Sanger-based sequencing.” He would spend much of the next two decades Vol. 55 | No. 5 | 2013
trying to find a less costly alternative. And he wasn’t the only one. Jonathan Rothberg ran his first sequencing reaction as an undergrad at Carnegie Mellon University in 1983. He entered Yale University as a doctoral student two years later, and by the time he graduated in 1991 had managed to completely sequence a neuronal gene called slit—9000 bases decoded in six years. Despite the slow pace, it was apparent even then that the burgeoning field of genomics promised to transform medical science, and in 1991 Rothberg founded CuraGen, a biotech firm devoted to mining the human genome for drug targets and insights into complex diseases. By 1999, CuraGen was a $5 billion, publicly traded company, and Rothberg was its CEO. “I thought I was on top of the world.”
Jonathan Rothberg as an undergraduate at Carnegie Mellon University. Credit: Jonathan Rothberg
Then his son, Noah, was born and was having difficulty breathing. The “consensus” human genome that was being assembled at the time was of little immediate value for Noah. “I realized that I wasn’t as interested in the human genome; I was really interested in my son Noah’s genome. And that for me was my first introduction to personal genomics.” Up until then, genomics had taken what Rothberg calls a “Henry Ford approach” to sequencing, “where you just set up an existing technology, but you set it up as an assembly line.” That strategy, implemented at places like the Broad Institute in Cambridge, Massachusetts and the Sanger Institute in the UK, is difficult to scale to the point where sequencing an individual genome is financially and temporally practical. So Rothberg, like Church, was looking for a paradigm shift, which he found on the cover of a computer magazine that happened to be on his desk. That cover described a new Intel Pentium
227
chip. “We’ve been doing it wrong,” he thought. “It’s not about Henry Ford, it’s about [Intel co-founder] Gordon Moore.” What needed to happen was to scale not up but down. “We needed to miniaturize sequencing.” Over the next two weeks—the time allotted for his paternity leave—Rothberg hashed out a new design based on cloning by limiting dilution and a solid-phase version of pyrosequencing, a technique that at time was used only for calling SNPs. The result was the massively parallel sequencing-by-synthesis strategy that was the foundation for 454 Life Sciences. Church also realized the key to cheaper sequencing was miniaturization, and had spent much of the previous two decades testing and developing strategies to get there. One method called multiplex sequencing, which was developed in 1988, featured several characteristics of modern next-generation sequencing. DNA sequences were immobilized on a flat surface and then subjected to many alternating cycles of probing and capturing the data using a digital camera. Church had also developed a strategy in which tiny pools of identical molecules are created in situ by a polymerase—so-called polymerase-generated colonies, or “polonies.” “I try to familiarize myself with many different technologies,” Church says, describing himself as “constantly on the prowl for combinations that I could match up with sequencing human genomes, because clearly that cost was off by a million at least.” In 2005, Church and his team rolled his various ideas into a new sequencing-by-ligation strategy that they published in the journal Science (1). That same week, Rothberg’s team at 454 described their strategy in Nature (2). According to Rothberg, it takes more than good sequencing technology to produce a successful sequencing strategy. There’s also the ability to eliminate the DNA cloning steps that remain so critical in Sanger-based approaches. “You really have to crack both problems to make it into a successful technology,” he says. “Sequencing was half the problem, sample prep was the other.” Both groups solved the problem the same way: by making libraries on-the-fly on the surface of micron-sized beads, arraying and immobilizing them on a planar surface, and applying cycles of sequencing chemistry in situ. Then, instead of running gels or capillaries, they imaged the array over and over again, using computers to sort out the data. It’s a strategy that is now used in broad strokes by every major sequencing vendor on the market. Since Church and Rothberg’s 2005 publications, next-generation DNA sequencing methods have largely eclipsed Sanger sequencing, and www.BioTechniques.com
®
Sequencing-Ready DNA Libraries from 1 ng – 1 µg of DNA in 2 Hours or Less The NEXTflex™ Rapid DNA-Seq Kit offers sequencing-ready, Illuminacompatible libraries from 1 ng – 1 µg of DNA in two hours or less. Proven Enhanced Adapter Ligation Technology provides the largest number of unique sequencing reads available. With low input requirements, a streamlined library prep protocol and a cost-effective price point, the NEXTflex Rapid DNA-Seq Kit will help you meet your research goals faster than you ever thought possible.
Fast - Workflow in 2 hours or less Flexible - Input DNA from 1 ng to 1 µg Compatible - Ideal for clinical & FFPE samples Multiplexed - Up to 96 barcodes Available Now - Next day delivery
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Amplicon-Seq • DNA-Seq • Pre-Target Capture Bisulfite-Seq • Methyl-Seq • RNA-Seq Small RNA-Seq • Directional RNA-Seq PCR-Free DNA-Seq • ChIP-Seq • qRNA-Seq Rapid DNA-Seq • Multiple Platform Compatibility
Tech News genome-sequencing costs have dropped to nearly as constructing standard curves and tediously $1,000. Church’s ligation-based approach has titrating cycle numbers. This all changed in 1993. Russell Higuchi, been used or adapted by at least four commercial sequencing firms, including Life Technologies at Roche Molecular Systems, and colleagues and Complete Genomics, arguably the biggest described a method for real-time quantitative PCR sequencer of human genomes to date. Roche (qPCR). This method used an intercalating dye Diagnostics bought 454, and Rothberg went on to (ethidium bromide) and a CCD camera to record found Ion Torrent, whose semiconductor-based reaction progress as cycling proceeded, producing method the Broad Institute’s Chad Nusbaum has a reliably quantitative analysis of PCR amplification. Other methods soon followed. SYBR Green called “the first post-light” sequencing technology. For those who have been in the thick of supplanted ethidium, and other strategies were sequencing technology for its 30-plus year introduced, too, most notably Life Technologies’ history, there is today perhaps a sense of déjà popular TaqMan chemistry, which uses a fluorescently labeled oligonucleotide vu. Back in the 1970s, probe to assess target abundance, it was Sanger versus rather than a non-specific Maxam-Gilbert. Today, DNA-binding dye. fundamentally different Vrana became an early approaches are competing adopter. “I was bright enough in a crowded market— to see…the power with which it optical versus non-optical, could be used,” he says. Yet PCR sequencing-by-synthesis vs was not just about RNA quantisequencing-by-ligation, with tation. Vrana also used it for nanopores and other stratcreating mutants, genetic analysis, egies on the horizon. Like validation of microarray data, and VHS versus Betamax, there’s more. “It has turned out to be a a period in technology develbedrock in the field,” he says. opment when competing Today, qPCR is a hugely technologies can co-exist, but popular PCR variant, and RNA inevitably, the balance shifts. Only time will tell where it George Church in 1977. Credit: George quantification is among its most common applications. In 2008 will land. Church Vrana co-authored a review on PCR’s 25th anniversary that tracked the number of Making PCR Quantitative qPCR papers over time (3). The figure resembles 1983 was also a banner year for Kent Vrana. Vrana, the Elliot S. Vesell Professor and Chair of a typical qPCR amplification curve, he says. “They Pharmacology at Penn State College of Medicine could be superimposed on each other.” Of course, researchers knew long before the in Hershey, Pennsylvania (and a member of the BioTechniques editorial board), earned his PhD invention of qPCR that dyes such as ethidium that year. His thesis was biochemical, but in 1983, bromide can bind DNA. And they also knew about he says, “molecular biology was just breaking,” so PCR’s quantitative limitations. Why, then, did it take so long for someone to put the two together? he took a postdoc to learn the new technology. “It truly was an engineering solution,” he says. Moving on to West Virginia University, he started his own lab studying transcript abundance. “I don’t think people understood the potential for At the time, Vrana says, that meant Northern real-time quantitative PCR. To think back on it blotting. But the technique wasn’t easy. “Very labor now, the draconian things we used to do in terms intensive, very difficult to quantify, [and] we were of quantifying amplicons on ethidium bromide gels—I’m just stunned that we would do that.” using a lot of radioactivity to do this.” PCR, Vrana says, had an immediate impact. “It cut the time to analyze RNA down by probably a Breaking the Diffraction Barrier factor of five.” About the time that Higuchi was introducing But at the start, the technique wasn’t really the world to qPCR, Stefan Hell was preparing to quantitative. PCR, in its original form, was an upend the world of microscopy. endpoint assay, a plus/minus readout where data At the end of the 1980s, Hell was a doctoral are interpreted after the reaction has finished using student at the University of Heidelberg. Though gel electrophoresis. Band size indicates a positive his interests lay in more fundamental aspects of or negative reaction, yet band intensity has little physics, his thesis advisor directed him towards quantitative value because the reaction often isn’t an applied project, studying the application of linear. To circumvent that problem, researchers confocal microscopy to inspect photolithographic like Vrana had to take extreme measures, such structures. It wasn’t where Hell wanted to go.
228
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TAKE TAKE ACTION TAKE TAKEACTION ACTION ACTION TAKE TAKE ACTION ACTION AGAINST AGAINST AGAINST AGAINST TAKE TAKE ACTION ACTION AGAINST AGAINST CONTAMINATION CONTAMINATION CONTAMINATION CONTAMINATION AGAINST AGAINST CONTAMINATION CONTAMINATION WITH WITH WITH WITH CONTAMINATION CONTAMINATION ® ®® WITH WITH QMI QMI SAFE-SEPTUM QMI QMI®®®®®®SAFE-SEPTUM SAFE-SEPTUM SAFE-SEPTUM WITH WITH QMI QMI®®®SAFE-SEPTUM SAFE-SEPTUM Sample, Sample, Sample, Inoculate Inoculate Or Or OrAdd Add AddNutrients Nutrients Nutrients QMIInoculate SAFE-SEPTUM SAFE-SEPTUM QMI Sample, Sample, Sample,Inoculate Inoculate InoculateOr Or OrAdd Add AddNutrients Nutrients Nutrients
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651-501-2337 651-501-2337 651-501-2337 651-501-2337 651-501-2337 651-501-2337 Email: Email: Email:[email protected] [email protected] [email protected] 651-501-2337 651-501-2337 651-501-2337 Email: Email: Email:[email protected] [email protected] [email protected] 651-501-2337 651-501-2337 651-501-2337 Email: Email: Email:[email protected] [email protected] [email protected] Email: Email: Email: [email protected] [email protected] [email protected] www.qmisystems.com www.qmisystems.com
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TAKE ACTION AGAINST CONTAMINATION WITH Tech® News QMI SAFE-SEPTUM “After a while, I became kind of frustrated, excitation beam activates all the fluorophores in a and I was thinking about what could you do diffraction-limited spot under the microscope. A Sample, Inoculate Or else Add Nutrients with light microscopy,” Hell, now Director of the second stimulated emission laser overlaid on the Your Bioreactor Aseptically MaxTo Planck Institute for Biophysical Chemistry in! excitation beam causes fluorophores in the overlap region to stay dark. By altering the intensity of the Göttingen, Germany, recalls. As he contemplated the fundamentals of second laser, he could raise or lower the resolution, light microscopy, Hell began to think about the theoretically down to the size of a single molecule. so-called diffraction (or Abbe) limit, which says “You can break the diffraction barrier without two objects cannot be distinguished under a having to break diffraction,” he says. light microscope if they are closer than half the The result was the first so-called super-resolution technique. Alternative strategies followed wavelength of light used to illuminate them. That limitation is a function of the properties a few years later, especially PALM and STORM, of light and lenses, and since the invention of the as well as another Hell-lab invention, RESOLFT, microscope it has been practically inviolable. And which derives multiple states from the cis-trans indeed, it is inviolable—the wave properties of isomerization of fluorescent molecules. Regardless of their acronyms, these techniques light mean optics can only be so precise and can focus only so sharply. But that doesn’t mean it isn’t all rely on the same fundamental phenomenon, says Hell, the ability of fluorophores to assume two possible to make the limit irrelevant. different states, making adjacent nanoscale features distinguishable. “The on/off transition, the fact that not all the molecules residing within a 200-nm zone are capable of sending light back, this has been the essential element for overcoming the diffraction barrier,” Hell says. Super-resolution microscopy is having “a lasting impact” on the life sciences and basic biomedical research. But the power of two-state transitions isn’t yet played out. Other molecules can adopt multiple states, too, including Ramanactive probes. “I’m totally convinced that future generations will not see the light microscope just from a wave perspective, but also from a state perspective,” he Stefan Hell, circa 1994. Credit: Pekka Hänninen says, a totally different perspective from when he Hell started exploring “molecular transitions,” started thinking about the problem in 1983. the ability of fluorescent molecules to occupy one of two states, active or silent. Consulting textbooks References on everything from quantum optics to nuclear 1. M. Margulies et al. 2005. Genome sequencing physics, he noticed a phenomenon he had learned in open microfabricated high density picoliter The QMI Safe-Septum is: about as a first-year student: stimulated emission. reactors. Nature, 437:376–80. “If you realize that you want to keep things 2. J. Shendure et al. 2005. Accurate multiplex dark, if you’re trained as a physicist, then you know polony sequencing of an evolved bacterial Aseptic the most fundamental way of turning a fluorogenome. Science, 309:1728–32, Sept. 9, 2005. & Temperature phorePressure off is to instantly send it down to theSafe ground 3. H.D. VanGuilder, K.E. Vrana, and W.M. state Pre-Sterilized by [using] a beam inducing stimulated Freeman. 2008. Twenty-five years of quantiemission,” he says. tative PCR for gene expression analysis. Easy TooutRetrofit Hell worked the mathematics of this idea— BioTechniques, 44[5]:619 - 26. whichValidated he now calls “the on/off game”—in 1993 and 4. T.A. Klar, S.W. Hell. 1999. Subdiffraction published his proposal in 1994. He then spent the resolution in far-field fluorescence microscopy. next half-decade trying to convince the scientific Optics Letters, 24[14]:954 - 6. world (and funding agencies) that he was right. He 5. T.A. Klar et al. 2000. Fluorescence microscopy finally got the money and lab space to implement with diffraction resolution barrier broken by stimuhis idea in 1998, and demonstrated the technique lated emission. Proc. Natl. Acad. Sci., 97:8206-10. works in a pair of papers in 1999 and 2000 (4,5). The technique he invented is called STED Written by Jeffrey Perkel, Ph.D. (stimulated emission depletion) microscopy and uses two lasers to improve resolution from BioTechniques 55:227-230 (November 2013) about 200 nm651-501-2337 to 50 nm. The sharply focused doi: 10.2144/000114103
Email: [email protected] 230
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Sequencing: Moving Up By Scaling Down In 1983 George Church was nearing the end of his graduate work at Harvard University. He had matriculated in 1977, and that same year joined the lab of DNA sequencing pioneer (and future Nobel laureate) Walter Gilbert. From the beginning, Church was focused on genome sequencing. In 1974, while still a Duke University undergraduate, he manually keyed in and folded the structure of every then-known tRNA sequence. “I started hoping that every person could afford to see their own DNA sequence,” he writes on his web site. Graduating in 1984, his thesis described “the first methods for direct genome sequencing, molecular multiplexing [and] barcoding.” That year, Church participated in a meeting at Alta, Utah that would launch the Human Genome Project (HGP). At the time, the only technology capable of decoding the human genome was Sanger dideoxy sequencing, and as the HGP took shape, some argued that the way forward was to ramp up existing technology. But Church argued for a different solution. “I felt that $3 billion was an unacceptable amount of money for Sanger-based sequencing.” He would spend much of the next two decades Vol. 55 | No. 5 | 2013
trying to find a less costly alternative. And he wasn’t the only one. Jonathan Rothberg ran his first sequencing reaction as an undergrad at Carnegie Mellon University in 1983. He entered Yale University as a doctoral student two years later, and by the time he graduated in 1991 had managed to completely sequence a neuronal gene called slit—9000 bases decoded in six years. Despite the slow pace, it was apparent even then that the burgeoning field of genomics promised to transform medical science, and in 1991 Rothberg founded CuraGen, a biotech firm devoted to mining the human genome for drug targets and insights into complex diseases. By 1999, CuraGen was a $5 billion, publicly traded company, and Rothberg was its CEO. “I thought I was on top of the world.”
Jonathan Rothberg as an undergraduate at Carnegie Mellon University. Credit: Jonathan Rothberg
Then his son, Noah, was born and was having difficulty breathing. The “consensus” human genome that was being assembled at the time was of little immediate value for Noah. “I realized that I wasn’t as interested in the human genome; I was really interested in my son Noah’s genome. And that for me was my first introduction to personal genomics.” Up until then, genomics had taken what Rothberg calls a “Henry Ford approach” to sequencing, “where you just set up an existing technology, but you set it up as an assembly line.” That strategy, implemented at places like the Broad Institute in Cambridge, Massachusetts and the Sanger Institute in the UK, is difficult to scale to the point where sequencing an individual genome is financially and temporally practical. So Rothberg, like Church, was looking for a paradigm shift, which he found on the cover of a computer magazine that happened to be on his desk. That cover described a new Intel Pentium
227
chip. “We’ve been doing it wrong,” he thought. “It’s not about Henry Ford, it’s about [Intel co-founder] Gordon Moore.” What needed to happen was to scale not up but down. “We needed to miniaturize sequencing.” Over the next two weeks—the time allotted for his paternity leave—Rothberg hashed out a new design based on cloning by limiting dilution and a solid-phase version of pyrosequencing, a technique that at time was used only for calling SNPs. The result was the massively parallel sequencing-by-synthesis strategy that was the foundation for 454 Life Sciences. Church also realized the key to cheaper sequencing was miniaturization, and had spent much of the previous two decades testing and developing strategies to get there. One method called multiplex sequencing, which was developed in 1988, featured several characteristics of modern next-generation sequencing. DNA sequences were immobilized on a flat surface and then subjected to many alternating cycles of probing and capturing the data using a digital camera. Church had also developed a strategy in which tiny pools of identical molecules are created in situ by a polymerase—so-called polymerase-generated colonies, or “polonies.” “I try to familiarize myself with many different technologies,” Church says, describing himself as “constantly on the prowl for combinations that I could match up with sequencing human genomes, because clearly that cost was off by a million at least.” In 2005, Church and his team rolled his various ideas into a new sequencing-by-ligation strategy that they published in the journal Science (1). That same week, Rothberg’s team at 454 described their strategy in Nature (2). According to Rothberg, it takes more than good sequencing technology to produce a successful sequencing strategy. There’s also the ability to eliminate the DNA cloning steps that remain so critical in Sanger-based approaches. “You really have to crack both problems to make it into a successful technology,” he says. “Sequencing was half the problem, sample prep was the other.” Both groups solved the problem the same way: by making libraries on-the-fly on the surface of micron-sized beads, arraying and immobilizing them on a planar surface, and applying cycles of sequencing chemistry in situ. Then, instead of running gels or capillaries, they imaged the array over and over again, using computers to sort out the data. It’s a strategy that is now used in broad strokes by every major sequencing vendor on the market. Since Church and Rothberg’s 2005 publications, next-generation DNA sequencing methods have largely eclipsed Sanger sequencing, and www.BioTechniques.com
®
Sequencing-Ready DNA Libraries from 1 ng – 1 µg of DNA in 2 Hours or Less The NEXTflex™ Rapid DNA-Seq Kit offers sequencing-ready, Illuminacompatible libraries from 1 ng – 1 µg of DNA in two hours or less. Proven Enhanced Adapter Ligation Technology provides the largest number of unique sequencing reads available. With low input requirements, a streamlined library prep protocol and a cost-effective price point, the NEXTflex Rapid DNA-Seq Kit will help you meet your research goals faster than you ever thought possible.
Fast - Workflow in 2 hours or less Flexible - Input DNA from 1 ng to 1 µg Compatible - Ideal for clinical & FFPE samples Multiplexed - Up to 96 barcodes Available Now - Next day delivery
Visit BiooNGS.com to accelerate your library prep.
Amplicon-Seq • DNA-Seq • Pre-Target Capture Bisulfite-Seq • Methyl-Seq • RNA-Seq Small RNA-Seq • Directional RNA-Seq PCR-Free DNA-Seq • ChIP-Seq • qRNA-Seq Rapid DNA-Seq • Multiple Platform Compatibility
Tech News genome-sequencing costs have dropped to nearly as constructing standard curves and tediously $1,000. Church’s ligation-based approach has titrating cycle numbers. This all changed in 1993. Russell Higuchi, been used or adapted by at least four commercial sequencing firms, including Life Technologies at Roche Molecular Systems, and colleagues and Complete Genomics, arguably the biggest described a method for real-time quantitative PCR sequencer of human genomes to date. Roche (qPCR). This method used an intercalating dye Diagnostics bought 454, and Rothberg went on to (ethidium bromide) and a CCD camera to record found Ion Torrent, whose semiconductor-based reaction progress as cycling proceeded, producing method the Broad Institute’s Chad Nusbaum has a reliably quantitative analysis of PCR amplification. Other methods soon followed. SYBR Green called “the first post-light” sequencing technology. For those who have been in the thick of supplanted ethidium, and other strategies were sequencing technology for its 30-plus year introduced, too, most notably Life Technologies’ history, there is today perhaps a sense of déjà popular TaqMan chemistry, which uses a fluorescently labeled oligonucleotide vu. Back in the 1970s, probe to assess target abundance, it was Sanger versus rather than a non-specific Maxam-Gilbert. Today, DNA-binding dye. fundamentally different Vrana became an early approaches are competing adopter. “I was bright enough in a crowded market— to see…the power with which it optical versus non-optical, could be used,” he says. Yet PCR sequencing-by-synthesis vs was not just about RNA quantisequencing-by-ligation, with tation. Vrana also used it for nanopores and other stratcreating mutants, genetic analysis, egies on the horizon. Like validation of microarray data, and VHS versus Betamax, there’s more. “It has turned out to be a a period in technology develbedrock in the field,” he says. opment when competing Today, qPCR is a hugely technologies can co-exist, but popular PCR variant, and RNA inevitably, the balance shifts. Only time will tell where it George Church in 1977. Credit: George quantification is among its most common applications. In 2008 will land. Church Vrana co-authored a review on PCR’s 25th anniversary that tracked the number of Making PCR Quantitative qPCR papers over time (3). The figure resembles 1983 was also a banner year for Kent Vrana. Vrana, the Elliot S. Vesell Professor and Chair of a typical qPCR amplification curve, he says. “They Pharmacology at Penn State College of Medicine could be superimposed on each other.” Of course, researchers knew long before the in Hershey, Pennsylvania (and a member of the BioTechniques editorial board), earned his PhD invention of qPCR that dyes such as ethidium that year. His thesis was biochemical, but in 1983, bromide can bind DNA. And they also knew about he says, “molecular biology was just breaking,” so PCR’s quantitative limitations. Why, then, did it take so long for someone to put the two together? he took a postdoc to learn the new technology. “It truly was an engineering solution,” he says. Moving on to West Virginia University, he started his own lab studying transcript abundance. “I don’t think people understood the potential for At the time, Vrana says, that meant Northern real-time quantitative PCR. To think back on it blotting. But the technique wasn’t easy. “Very labor now, the draconian things we used to do in terms intensive, very difficult to quantify, [and] we were of quantifying amplicons on ethidium bromide gels—I’m just stunned that we would do that.” using a lot of radioactivity to do this.” PCR, Vrana says, had an immediate impact. “It cut the time to analyze RNA down by probably a Breaking the Diffraction Barrier factor of five.” About the time that Higuchi was introducing But at the start, the technique wasn’t really the world to qPCR, Stefan Hell was preparing to quantitative. PCR, in its original form, was an upend the world of microscopy. endpoint assay, a plus/minus readout where data At the end of the 1980s, Hell was a doctoral are interpreted after the reaction has finished using student at the University of Heidelberg. Though gel electrophoresis. Band size indicates a positive his interests lay in more fundamental aspects of or negative reaction, yet band intensity has little physics, his thesis advisor directed him towards quantitative value because the reaction often isn’t an applied project, studying the application of linear. To circumvent that problem, researchers confocal microscopy to inspect photolithographic like Vrana had to take extreme measures, such structures. It wasn’t where Hell wanted to go.
228
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TAKE TAKE ACTION TAKE TAKEACTION ACTION ACTION TAKE TAKE ACTION ACTION AGAINST AGAINST AGAINST AGAINST TAKE TAKE ACTION ACTION AGAINST AGAINST CONTAMINATION CONTAMINATION CONTAMINATION CONTAMINATION AGAINST AGAINST CONTAMINATION CONTAMINATION WITH WITH WITH WITH CONTAMINATION CONTAMINATION ® ®® WITH WITH QMI QMI SAFE-SEPTUM QMI QMI®®®®®®SAFE-SEPTUM SAFE-SEPTUM SAFE-SEPTUM WITH WITH QMI QMI®®®SAFE-SEPTUM SAFE-SEPTUM Sample, Sample, Sample, Inoculate Inoculate Or Or OrAdd Add AddNutrients Nutrients Nutrients QMIInoculate SAFE-SEPTUM SAFE-SEPTUM QMI Sample, Sample, Sample,Inoculate Inoculate InoculateOr Or OrAdd Add AddNutrients Nutrients Nutrients
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The The TheQMI QMI QMISafe-Septum Safe-Septum Safe-Septumis: is:is: The The TheQMI QMI QMISafe-Septum Safe-Septum Safe-Septumis: is:is: The The TheQMI QMI QMISafe-Septum Safe-Septum Safe-Septumis: is:is: Aseptic Aseptic Aseptic The The The QMI QMI QMI Safe-Septum Safe-Septum Safe-Septum is: is:is: Aseptic Aseptic Aseptic Pressure Pressure Pressure & & & Temperature Temperature Temperature Safe Safe Safe Aseptic Aseptic Aseptic Pressure Pressure Pressure& &&Temperature Temperature TemperatureSafe Safe Safe Pre-Sterilized Pre-Sterilized Pre-Sterilized Aseptic Aseptic Aseptic Pressure Pressure Pressure& &&Temperature Temperature TemperatureSafe Safe Safe Pre-Sterilized Pre-Sterilized Pre-Sterilized Easy Easy Easy To To ToRetrofit Retrofit Retrofit Pressure Pressure Pressure & & & Temperature Temperature Temperature Safe Safe Safe Pre-Sterilized Pre-Sterilized Pre-Sterilized Easy Easy EasyTo To ToRetrofit Retrofit Retrofit Validated Validated Validated Pre-Sterilized Pre-Sterilized Pre-Sterilized Easy Easy EasyTo To ToRetrofit Retrofit Retrofit Validated Validated Validated Easy Easy Easy To To ToRetrofit Retrofit Retrofit Validated Validated Validated Validated Validated Validated
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TAKE ACTION AGAINST CONTAMINATION WITH Tech® News QMI SAFE-SEPTUM “After a while, I became kind of frustrated, excitation beam activates all the fluorophores in a and I was thinking about what could you do diffraction-limited spot under the microscope. A Sample, Inoculate Or else Add Nutrients with light microscopy,” Hell, now Director of the second stimulated emission laser overlaid on the Your Bioreactor Aseptically MaxTo Planck Institute for Biophysical Chemistry in! excitation beam causes fluorophores in the overlap region to stay dark. By altering the intensity of the Göttingen, Germany, recalls. As he contemplated the fundamentals of second laser, he could raise or lower the resolution, light microscopy, Hell began to think about the theoretically down to the size of a single molecule. so-called diffraction (or Abbe) limit, which says “You can break the diffraction barrier without two objects cannot be distinguished under a having to break diffraction,” he says. light microscope if they are closer than half the The result was the first so-called super-resolution technique. Alternative strategies followed wavelength of light used to illuminate them. That limitation is a function of the properties a few years later, especially PALM and STORM, of light and lenses, and since the invention of the as well as another Hell-lab invention, RESOLFT, microscope it has been practically inviolable. And which derives multiple states from the cis-trans indeed, it is inviolable—the wave properties of isomerization of fluorescent molecules. Regardless of their acronyms, these techniques light mean optics can only be so precise and can focus only so sharply. But that doesn’t mean it isn’t all rely on the same fundamental phenomenon, says Hell, the ability of fluorophores to assume two possible to make the limit irrelevant. different states, making adjacent nanoscale features distinguishable. “The on/off transition, the fact that not all the molecules residing within a 200-nm zone are capable of sending light back, this has been the essential element for overcoming the diffraction barrier,” Hell says. Super-resolution microscopy is having “a lasting impact” on the life sciences and basic biomedical research. But the power of two-state transitions isn’t yet played out. Other molecules can adopt multiple states, too, including Ramanactive probes. “I’m totally convinced that future generations will not see the light microscope just from a wave perspective, but also from a state perspective,” he Stefan Hell, circa 1994. Credit: Pekka Hänninen says, a totally different perspective from when he Hell started exploring “molecular transitions,” started thinking about the problem in 1983. the ability of fluorescent molecules to occupy one of two states, active or silent. Consulting textbooks References on everything from quantum optics to nuclear 1. M. Margulies et al. 2005. Genome sequencing physics, he noticed a phenomenon he had learned in open microfabricated high density picoliter The QMI Safe-Septum is: about as a first-year student: stimulated emission. reactors. Nature, 437:376–80. “If you realize that you want to keep things 2. J. Shendure et al. 2005. Accurate multiplex dark, if you’re trained as a physicist, then you know polony sequencing of an evolved bacterial Aseptic the most fundamental way of turning a fluorogenome. Science, 309:1728–32, Sept. 9, 2005. & Temperature phorePressure off is to instantly send it down to theSafe ground 3. H.D. VanGuilder, K.E. Vrana, and W.M. state Pre-Sterilized by [using] a beam inducing stimulated Freeman. 2008. Twenty-five years of quantiemission,” he says. tative PCR for gene expression analysis. Easy TooutRetrofit Hell worked the mathematics of this idea— BioTechniques, 44[5]:619 - 26. whichValidated he now calls “the on/off game”—in 1993 and 4. T.A. Klar, S.W. Hell. 1999. Subdiffraction published his proposal in 1994. He then spent the resolution in far-field fluorescence microscopy. next half-decade trying to convince the scientific Optics Letters, 24[14]:954 - 6. world (and funding agencies) that he was right. He 5. T.A. Klar et al. 2000. Fluorescence microscopy finally got the money and lab space to implement with diffraction resolution barrier broken by stimuhis idea in 1998, and demonstrated the technique lated emission. Proc. Natl. Acad. Sci., 97:8206-10. works in a pair of papers in 1999 and 2000 (4,5). The technique he invented is called STED Written by Jeffrey Perkel, Ph.D. (stimulated emission depletion) microscopy and uses two lasers to improve resolution from BioTechniques 55:227-230 (November 2013) about 200 nm651-501-2337 to 50 nm. The sharply focused doi: 10.2144/000114103
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