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Insight & Intelligence : Feb 21, 2013
Isothermal PCR Gets Off The Dime
Two companies aim to make PCR and its need for thermal cycling instrumentation obsolete.!--h2>
Last year, EnviroLogix and Douglas Scientific signed a collaborative agreement to develop and optimize technology that they say will enable high-throughput real-time and endpoint nucleic acid analysis.
Put another way, the companies intend to make polymerase chain reaction (PCR) and its need for thermal cycling instrumentation obsolete by leveraging EnviroLogix isothermal DNAble chemistry and Douglas’ Array Tape™ platform into a novel high-throughput platform for fast PCR.
PCR has long been the method of choice for scientists wishing to make sufficient amounts of identical genetic material for study and analysis. Applications for PCR include genotyping, SNP analysis, drug target validation, and quantitative gene expression analysis, which require quantification of the amount of DNA in a starting sample.
And to get results in real time, researchers use real-time PCR, or quantitative PCR (qPCR) that monitors PCR amplification as it occurs. qPCR can also be combined with reverse transcription (RT-qPCR) to quantify the amount of RNA in a starting sample.
How PCR Works
Closely patterned after the natural DNA replication cycle that occurs in cells, each PCR cycle involves a denaturation, annealing, and extension step in a programmed thermal cycler. The cycler automatically controls and alternates the temperatures for each stage of the reaction, raising and lowering the temperature of the chemical components at specific times and for a preset number of cycles.
Two factors crucial in enabling PCR to be used as an efficient laboratory technique were the discovery of thermostable DNA polymerases, derived from thermophilic bacterium (Thermus aquaticus) and the invention of the automated thermal cycler. Unlike regular DNA polymerases, T. aquaticus or Taq polymerases can withstand heating to 95°C meaning they do not need to be added at each new PCR cycle. Using Taq polymerase, the extension step can be performed at 72°C.
Today’s modern thermocyclers comprise metal blocks where tubes containing the PCR reaction can be inserted. The thermocycler is pre-programmed to move between temperatures for highly accurate and fast cycling, enabling most PCR reactions to be completed in less than two hours.
PCR is now the most widely used technique in molecular biology, biochemistry, and medicine, with the market growing at an annual rate of over 10%. By 2015, the global market is expected to surpass the $38 billion mark.
Currrently, there are more than 50 PCR techniques in use, with qPCR, RT-PCR, Hot Start, Multiplex, and Taqman techniques having the market lead, and expected to see continued growth.
But none of these change the basic PCR paradigm, which remains expensive, time-consuming, instrument and reagent-intensive, and requires extensive sample preparation. And high throughput for real-time PCR assays has been hard to achieve because of the relatively long reaction times required for the PCR cycle of denaturing, extension, and reannealing.
Alternatives to PCR
Isothermal amplification technology, or DNA amplification without the need for a thermal cycler, has potentially offered a cheaper, much faster, less reagent-intensive, and more adaptable method than PCR. These include methods based on, for example, strand displacement amplification, nucleic acid sequence-based amplification, self-sustained sequence replication, rolling circle amplification, loop-mediated amplification, and helicase-dependent amplification.
Scientists say while these methods may overcome some of the technical and cost barriers associated with PCR and do provide amplification of target sequences equal to PCR, they may require complex protocols, the use of multiple enzymes and/or special reagents and, in particular, either a high-temperature denaturation step or an enzyme-based method for promoting nicking and strand displacement.
But according to Douglas Scientific (manufacturer of the Array Tape platform) and its partner, EnviroLogix (which provides its proprietary DNAble novel DNA amplification chemistry), combining these technologies into an automated solution in one platform could represent a new paradigm in real-time nucleic acid analysis.
DNAble, EnviroLogix says, is a very rapid, highly specific isothermal nucleic acid amplification technology that can amplify both RNA and DNA targets with single-base resolution a billion-fold in 5–15 minutes at a single and constant temperature. It features EnviroLogix’ DNAble v2.0 chemistry, which reportedly produces highly specific, rapid, multiplexed, quantitative results rivaling qPCR. Unlike qPCR, which requires purified DNA, the company says DNAble can amplify a target sequence from a crude sample preparation and do so with minimal equipment.
DNAble works by using a nicking enzyme and a strand-displacing polymerase to generate small pieces of DNA that feed a DNA extension reaction; it consists of alternating cycles of nicking and extension processes, leading to exponential amplification. This is much faster, the company says, than the time-consuming thermal cycling that relies on costly equipment. But in contrast to other isothermal methods that rely on DNA strand nicking and displacement synthesis, DNAble chemistry v2.0 has eliminated off-target amplification of nonspecific products.
Further, the company said, DNAble shows little inhibitory effects from a crude extraction or exogenous DNA. As a result, DNAble can reportedly allow for highly sensitive detection with minimal to no sample preparation. The company says its data confirms the high specificity of the assay with no cross-reactivity to closely related bacteria or synthetic mismatch targets.
Lars Erik Peters, Envirologix’ director of product development for molecular diagnostics, told GEN, “Isothermal methods are prone to amplify huge haystacks of nonspecific byproducts but finding the needles of specific product has been difficult. You do not have the high temperature control that you have with PCR to control specificity.” He explained that EnviroLogix “set out to make isothermal technology into a process that is directly comparable to PCR regarding assay design and simplicity of use. This was a real technology endeavor because it not only required optimization of chemistry, but also development of assay design software and instrumentation.”
He further explained that the “background noise in isothermal was extraordinary. But now we have solved the background generation problem and have data that looks like PCR data.”
And Douglas Scientific’s president and COO Dan Malmstrom said, “When we delivered Array Tape for PCR we increased throughput by 10X, reduced reagent costs up to 80–90%, and offered an automated solution that saved laboratories huge amounts of money. We are taking it to the next level—this is a game changer.”
Both companies say that primary targets for their platform are the agbio and plant genomics markets, where they say they already have a good footprint. “This is only possible,” they said, “because in contrast to PCR the technology works with crude extracts, and DNA preparation for PCR has often been more expensive than the PCR reactions themselves.”
Whether this technology produces the anticipated revolution in DNA amplification and extends its applicability remains to be seen.
Patricia Fitzpatrick Dimond, Ph.D. ([email protected]), is technical editor at Genetic Engineering & Biotechnology News.
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