[ 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 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.

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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 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

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“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 . 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 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. As was critical. The discovery and use of the PCR technique has evolved.

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The LightCycler 1.5 real-time PCR APBN: Tell us about your APBN: Tell us how you came system was the first to monitor the development of rapid-cycle to invent the LightCycler hybridization process through melting PCR techniques. system. Why did you choose curve analysis and automate absolute Carl Wittwer: When we started to focus on Real-Time PCR? quantification. The LightCycler 2.0 work on PCR, commercial thermal Carl Wittwer: We were doing a lot system advanced multicolor analysis cyclers were not available. We built of rapid cycle PCR in capillaries to provide researchers with up to a device based on heating with a hair — optically clear glass capillaries. My six colors of analysis. As real-time dryer to amplify samples in capillary clinical job at the time was to manage PCR evolved and high throughput tubes. We soon realized that the air/ a flow cytometry laboratory, where analysis became more important, capillary system could be driven very the fluorescence of cells is commonly Roche developed the LightCycler quickly, and set out to establish the measured in a fluid stream. Instead of 480 system to accommodate both temperature and time requirements measuring the fluorescence of cells, 96 and 384-well microtiter plates. for PCR. With nimble control of we asked, why not monitor PCR The variable plate formats available temperature, we established that solutions in glass capillaries during on the LightCycler 480 meant that denaturation and annealing times, PCR? Russ Higuchi had already scientists could exchange thermal once reached, did not need to be held, demonstrated real-time monitoring block cyclers in a few minutes that is, “0” second temperature spikes with ethidium bromide, and David without having to recalibrate the were entirely adequate for PCR Gelfand had suggested that the instrument. and 30 cycles could be completed activity of Taq might be in 10-30 minutes. Indeed, short used to generate probe fluorescence APBN: Tell us about your denaturation times improved yield in a closed tube. The capillaries could research background and short annealing times improved be placed within the temperature and how it led to your specificity. The only reason to hold cycling chamber on a carousel and current focus in medical denaturation and annealing times rotated into position for optical diagnostics. was to let the thermal cycler catch interrogation. We obtained funding up and equilibrate all samples to the through a hybrid academic/small Carl Wittwer: I am an analytical target temperatures. business grant to combine rapid air biochemist trained in enzymology Rapid cycle PCR was developed cycling with fluorescent monitoring with an interest in developing clinical in the early 1990s and found during PCR. The fluorescence optics applications. After medical school and commercial use in the carousel on the LightCycler prototype were a residency in pathology, I directed a LightCyclers (versions 1.5 and obtained from an obsolete flow flow cytometry laboratory as junior 2.0). Plate-based systems such as cytometer. Curiously enough, we faculty at the University of Utah the LightCycler 480 are getting never got the exonuclease method and new technology development at faster and there is renewed interest to work, so we moved on to ARUP, a national reference laboratory in “FAST” PCR, but the 10-30 develop dual hybridization probes in the USA. My first new technology minute criteria of rapid cycling is (HybProbes®), SYBR® Green I and focus was PCR in the late 1980s. The seldom achieved on plate-based melting analysis. 1988 Science paper introducing Taq systems. Rapid cycling is widely used Why real-time PCR? In graduate polymerase had just appeared and it on microfluidic platforms where school I used to stand by the printer was clear PCR was destined to have the small sample volumes lend of a scintillation counter so that clinical applications. themselves to rapid cycling. I could plot the results of a radio immuno assay (RIA) assay as it came off of the instrument. Turn around time has always been important in

www.asiabiotech.com Volume 13 > Number 4 > 2009 ■ 33 [ Feature ] my research. I would much rather next response is usually, “Who would not detect heteroduplexes well. get a few good results rapidly than want to do that?” In the mid 1990s I have to wait longer for a large remember Russ Higuchi telling me APBN: Tell us about some batch of results that might all be I was one of the very few scientists off-shoots of PCR. compromised. By making real-time interested in real-time PCR — the PCR fast and displaying results each commercial and scientific potential Carl Wittwer: Over the years, many cycle, many scientists now enjoy was not realized for many years. variants of PCR have been developed. watching real-time PCR in “real- I have been fortunate enough to One of them is quantitative real-time time”. follow this process through three PCR, more commonly known as technologies, rapid PCR cycling, qPCR. With qPCR, the exponential the LightCycler system, and high phase of PCR is monitored, allowing APBN: What were some of resolution melting analysis. determination of the initial amount the challenges you faced in of target. developing the LightCycler Roche Diagnostics provides system? APBN: Most DNA research laboratories all over the sequencing methods Carl Wittwer: The engineering world with a series of different take the electrophoresis of the instruments relied heavily real-time PCR systems to suit their approach at some point. on existing tools and there were needs. With the LightCycler 2.0 How does High-Resolution many contributions from hardware and LightCycler 480 real-time PCR Melting Analysis compare and software developers at Idaho systems, researchers have access to to these other sequencing Technology and Roche Applied both rapid carousel-based and high- methods? Science. One recurring challenge throughput systems. This year, we was to reduce the initial prototypes Cart Wittwer: High resolution celebrate the 10th anniversary of the to economically feasible commercial melting analysis does not sequence. LightCycler system. The LightCycler products. Because the fluorescence It can very rapidly re-sequence introduced many “firsts” to real-time generated during PCR was high, and determine sequence identity PCR, including rapid cycling, SYBR less inexpensive light sources and between different samples. In Green I, dual hybridization probes, detectors could be used instead of many cases this eliminates the need single hybridization probes, and lasers and photomultiplier tubes. for sequencing. The advantage of melting analysis. As a rapid, open The probes and melting high resolution melting is that no system, it remains the most user- analysis were challenging to develop processing is required. Just like real- friendly real-time instrument for because they were entirely new. time PCR, analysis remains closed- assay and technique development. The advantage we had was rapid tube. It has quickly been accepted The first genetic tests to receive turn around time. New methods as the mutation scanning method of FDA approval in the US (F5 and F2) are much easier to develop when choice for reasons of both accuracy were on the carousel LightCycler in multiple iterations are possible and ease of use. High resolution 2002, setting the standard for other within one working day. melting also provides very simple companies to follow. When a new method or solutions for genotyping. Different The extreme sensitivity of real- instrument is developed, the first instruments vary widely in their time PCR, with the ability to detect response is usually disbelief. I melting resolution. Many real-time even a single nucleic acid molecule, still remember a telephone call I instruments do not have either the makes it possible to detect fetal received in the early days of rapid resolution or software to perform high DNA in maternal serum for non- cycling, where I was told PCR was resolution melting. Heteroduplex invasive prenatal diagnosis. Digital not possible in 15 minutes. After detecting dyes (“saturation dyes”) are PCR is a recent modification where eventual scientific acceptance, the also necessary – SYBR Green I does the sample volume or concentration

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APBN: One of the reasons the ... a revolution similar to the enabling PCR method was invented, integration of Taq polymerase has not occurred was to address the problem of yet. One interesting development is a insufficient DNA for cloning, sequencing and recombinant thermostable that eliminates the studies. Are scientists working need for heat denaturation. with DNA today having that same problem? What other problems are being tackled is reduced so much so that each continue to research better reagents with PCR? reaction only contains one starting for PCR. Not surprisingly, the claims Carl Wittwer: Limited nucleic acid template. Emulsion PCR creates often exceed reality and a revolution continues to be a concern, whether millions of nanoliter PCRs within oil, similar to the enabling integration for single cell analysis, minute forensic a process that is central to most next- of Taq polymerase has not occurred samples, or extensive whole genome generation sequencing methods. yet. One interesting development is a studies. For DNA, pre-amplification thermostable helicase that eliminates or whole genome amplification the need for heat denaturation. APBN: The discovery of Taq methods provide a good solution. Polymerase revolutionized For RNA analysis, several RNA PCR and eventually led to APBN: What are your amplification methods have been the making of an automated thoughts on Helicase- developed. thermocycler. What are dependent amplification PCR methods continue to some newly discovered (HDA)? enable study of newly discovered nucleic acids that were unknown a enzymes, or biomolecules Carl Wittwer: By adding a helicase to few years ago. For example, PCR is that could trigger a similar separate the double helix of DNA, commonly used to study a new class revolution in PCR now? primer annealing and extension can of small RNA molecules that regulate occur without heat denaturation. Carl Wittwer: Advances have been , the microRNAs. As Under precise isothermal conditions, made in PCR reagents. These include another example, cell free nucleic similar amplification yields to PCR antibodies against the polymerase, acids have now been found in can be achieved. Isothermal reactions heat-labile chemical modifications saliva, urine, and blood plasma with such as HDA and loop mediated to the polymerase, primers or dNTPs, important clinical correlates. The amplification are alternatives to PCR and/or aptamers can be used to extreme sensitivity of PCR provides that do not require temperature improve specificity limiting extension a very powerful tool for biological cycling at the expense and at low temperatures before PCR has analysis. begun. Polymerases such as Tth complexity of additional reagents. can accept both DNA and RNA as Furthermore, isothermal techniques APBN: How do you envision templates, simplifying amplification are less metered than with PCR the future of PCR and the of RNA targets. Specialty polymerases temperature cycling, making precise LightCycler? with better heat stability, fidelity and quantification more challenging. polymerase speed have been created. Also, amplification is performed at Carl Wittwer: PCR has become a However, current PCR times are a constant temperature so melting basic tool in molecular biology. It limited by instrumentation, not the data cannot be extracted during will not go away. PCR and the human speed of the polymerase. amplification. genome project have “democratized” Many biotechnology companies molecular biology, enabling anyone

www.asiabiotech.com Volume 13 > Number 4 > 2009 ■ 35 [ Feature ] with a laboratory to investigate primers. High resolution melting will ambivalence are part of the process targets of their choice. Real-time expand into additional applications of innovation. No one will believe in and melting analysis expand the such as clone screening and sequence your method or instrument unless you do. Use the scientific literature to utility of PCR, making PCR analysis matching for transplantation expand awareness of your technology both easier and more powerful. The testing. by publishing applications that will LightCycler remains one of the interest other researchers. Work with most versatile tools available for the companies to develop your products, individual investigator. APBN: What advice do you and defer to them on all aspects Over time, PCR will become have for other researchers commercial. ■ even faster, more precise, and more developing molecular affordable with lower sample diagnostic techniques and volumes. For most applications, 5- instruments? 10 minute PCR is feasible. Labeled Carl Wittwer: R e m e m b e r probes will be replaced by unlabeled that rejection and commercial probes, either free or attached to

About Professor Carl Wittwer

Carl Wittwer is Professor of Pathology at the University of Utah Medical School. He is also the Medical Director of Flow Cytometry and the Advanced Technology Group at Associated Regional and University Pathologists (ARUP), and the Chief Science Officer of Idaho Technology. He received his PhD in Biochemistry from Utah State University and his MD from the University of Michigan. Dr. Wittwer has published more than 150 research articles and book chapters on technique and instrument development in molecular diagnostics. His focus is on simplifying DNA analysis so that separation steps, electrophoresis, and expensive probes are not required. Dr. Wittwer holds over 20 US patents and their foreign equivalents, mostly relating to the LightCycler system. He received small business innovation awards in 1999 and 2002 and was conferred the Governor of Utah Medal for Science and Technology in 2003, the AACC award for Outstanding Contributions to Clinical Chemistry in 2004, the IQLM Technical Advancement Award in 2005, the IFCC-Abbott Award for Significant Contributions to Molecular Diagnostics in 2005 and the AMP Award for Excellence in Molecular Diagnostics in 2008.

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