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The PCR Revolution – an Interview with Carl T Wittwer

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The PCR Revolution – an Interview with Carl T Wittwer [ Feature ] The PCR Revolution – An Interview with Carl T Wittwer by V. K. Sanjeed n the rapidly evolving field of biotechnology, few Itools have the privilege of becoming mainstays like polymerase chain reaction (PCR) – the technique used to generate millions of copies of a DNA sequence of interest. It has evolved into a ubiquitous and essential tool in almost every biology laboratory, since Kary Mullis developed the method in 1983. Few names stand out in the history of this molecular marvel, but Professor Carl T Wittwer, deserves special mention for his revolutionary LightCycler system. APBN speaks to the trail-blazer about the evolution of PCR and where he sees PCR progressing in the next decade. 30 ■ Volume 13 > Number 4 > 2009 www.asiabiotech.com [ Feature ] APBN: How did Kary Mullis too small to study, such as a strand of Although PCR can be performed first conceptualize the PCR hair or a drop of blood. in 10 to 15 minutes with the right technique in 1983? The PCR process is dramatized equipment, many samples are in the movie Jurassic Park, where often amplified together in array Carl Wittwer: In 1983, Kary Mullis scientists clone dinosaurs from the format in one to two hours. PCR is was working at Cetus Corporation blood of mosquitoes that were performed in a test tube, replicating as an oligonucleotide chemist when preserved in tree sap. A fragment the complicated process of DNA he first came up with the idea. of dinosaur DNA is copied by synthesis that usually occurs only According to him, the idea came two primers, a polymerase and inside cells. All that is required for to him late one night when he was temperature cycling of the reaction. PCR is the original DNA template, driving. He thought of using a pair of The high temperature (94°C) first commonly available reagents and a primers to bracket the desired DNA separates (or denatures) the DNA source of controlled heat. This is the sequence and to copy it using DNA double helix into two single strands. reason why PCR is such a simple yet polymerase – a technique which Cooling to a lower temperature elegant tool that has revolutionized would allow a small strand of DNA (55°C) allows two synthetic primers biology. to be copied many, many times. to find (or anneal to) the separated Cetus Corporation assigned Mullis strands. These primers are short to develop the PCR technique, and single strand DNA pieces that are APBN: What made PCR so he was able to produce definitive designed with a specific sequence popular amongst biologists, proof that it worked indeed in that targets the desired fragment. that it became an essential December 1983. These two primers anneal to opposite tool for almost every When first developed, there fragments of the separated DNA and biological lab? were complications with the PCR are pointed toward each other, so technique. First of all, the DNA Carl Wittwer: PCR is popular that at a third temperature (74°C), amongst biologists because polymerase used in each cycle a polymerase extends the primers to of replication was destroyed by it enables them to amplify and make double stranded copies of the duplicate genetic material, using a the high heat in the reaction and original target. This temperature had to be constantly replaced. very small amount of the original cycling is repeated over and over DNA template within a very short This increased the cost of the again and at each cycle, the amount process. The introduction of Taq period of time. PCR has made things of target DNA is doubled. more efficient for biologists when DNA polymerase in 1988 solved After 20 cycles, a million copies this problem. The Taq polymerase they are studying the genomes of of the desired DNA sequence have living organisms and examining the survived heating and thus only been made. Although it sounds needed to be added once in the underlying mechanisms that govern complicated, the primers can be how organisms work. Over the last beginning of the chain reaction. This designed, ordered and delivered made PCR much more affordable few years, scientists have been able to by overnight mail and all other map not just the human genome, but and efficient, making it much more components are common laboratory useful to researchers and scientists. even animal genomes, like the giant chemicals. Once biotechnology panda genome using PCR. suppliers realized what scientists Real-time PCR makes PCR APBN: In layman’s terms, needed for their PCR reactions, even simpler and more powerful. what is PCR and what they started supplying reagents Traditional PCR required additional makes it so simple and and primer-synthesizing services, steps to analyze the PCR products elegant? which made PCR that much more generated while real-time PCR convenient. Scientists specify which integrates analysis into the Carl Wittwer: PCR, or polymerase piece of DNA should be amplified amplification process. In the presence chain reaction, is widely used in with the primer sequences. Machines of fluorescent dyes or probes, PCR molecular biology, microbiology, developed for automatic temperature can be monitored by measuring the genetics, diagnostics and forensics. cycling in the late 1980s greatly fluorescence each cycle. This allows PCR makes it possible to study increased the speed and volume of quantification of the initial target, a samples of DNA that were previously PCR experiments. process now referred to as qPCR or www.asiabiotech.com Volume 13 > Number 4 > 2009 ■ 31 [ Feature ] “quantitative PCR”. With real-time magnesium is included in the reaction the heat-resistant Taq polymerase in PCR, not only do you determine in the form of MgCl2, inadequate 1988 greatly revolutionized the PCR the presence or absence of a target, thawing of the compound can result technique by eliminating the need for but how much is present. Examples in the formation of concentration additional polymerase. It also paved include mRNA quantification in gradients in the solution and result the way to automated temperature gene expression research and viral in a failed experiment. cycling without reagent addition. The load quantification in diagnostics. process of PCR suddenly became A further advancement beyond much simpler and more accessible. APBN: How has PCR evolved real-time PCR is to measure However, quantitative data was fluorescence continuously instead of to deal with the problem of still difficult to obtain, and the just once each cycle. By monitoring contamination? process remained qualitative until fluorescence as the temperature Carl Wittwer: Conventional PCR the introduction of real-time PCR changes, DNA hybridization can be requires additional analysis after instruments in the mid 1990s. followed as melting curves of PCR amplification, most commonly By monitoring fluorescence products or probes. The power separation on a gel or sequencing. in each cycle of PCR, the precise of melting analysis has advanced Such processing exposes PCR amount of starting template could recently as “high resolution melting products to the environment and be determined, allowing true analysis”, a method available on risks contamination of future PCR quantification. mRNA quantification the Roche LightCycler 480 that reactions with past amplification was critical for gene expression often eliminates the need for any products. However, real-time PCR research and viral load determination downstream analysis such as gel and melting analysis are “closed tube” in clinical applications. Time- separation or sequencing. processes, that is, the sample is never consuming and expensive microbial removed from the tube, even after diagnostics began to be replaced by faster, more accurate real-time PCR APBN: What are the amplification is complete. Since PCR assays. inherent weaknesses of products are never exposed to the environment, past reactions cannot The next advance was melting PCR? contaminate future reactions unless analysis – a process that goes beyond Carl Wittwer: Because PCR is such the “seal” is broken. conventional real-time PCR. In a sensitive test, poor or sloppy Prior to closed tube analysis, addition to monitoring fluorescence technique can cause original samples many laboratories adopted several once each cycle, fluorescence is to get contaminated. Also, common controls for contamination, including monitored continuously as the secondary structures known as physical separation of reaction set- temperature changes so that hairpins in DNA may result in up and post-PCR analysis. One of hybridization can be followed. the folding of DNA templates, the more popular ways to eliminate Both PCR product hybridization decreasing the yield of the product potential contaminating PCR with SYBR Green I and probe and even failure of the reaction. products is to incorporate dUTP for hybridization for genotyping can be Magnesium is also required dTTP and use uracil deglycosylase monitored at the end of PCR. As for PCR using the Taq polymerase. (UDG) to degrade synthesized melting technology became better Determining the optimum strands. and better, high resolution melting concentration to use is critical to analysis was introduced as the latest the success of the reaction, and APBN: What other factors method for product analysis. Labeled some components of the reaction probes were no longer necessary for mixture can reduce the amount of drove the evolution of PCR? genotyping and entire PCR products free magnesium, thus reducing the Carl Wittwer: The need for speed, could be scanned for single base activity of the enzyme. Excessive accuracy and cost-containment were magnesium present in the reaction, factors that have driven the evolution changes in only one copy of diploid on the other hand, can cause double of PCR. Initially, finding a polymerase DNA. stranded DNA and primers bound to that withstood the high temperatures The LightCycler real-time PCR incorrect template sites to stabilize, involved in the duplication of DNA systems are a good example of how reducing the product yield.
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