CORONA DEL MAR, Calif.—When Dr. Kary Banks Mullis’ good friend Ron Cook developed a machine that could produce oligonucleotides in two days, Mullis faced the prospect of firing the seven people in his lab who had been laboriously producing oligos manually. His only other option was increasing demand for the products. Soon Mullis was to visualize and experimentally validate the process that has become known as polymerase chain reaction (PCR)—which, in his own words, has increased the use of oligos a millionfold, and is used by “hundreds of companies that do it for a living.” Mission accomplished.
At the time, Mullis worked for Cetus Corp. in Emeryville, Calif., where no one was interested in helping him obtain the supply of purified thermophillic polymerase he needed—until two advisors, Hamilton Smith, a renowned microbiologist, and Ray Wu, Cornell’s acclaimed pioneer of genetic engineering, urged Cetus’ management to get someone on it. They did.
But not everything was yet coming up roses. In 1983, Mullis submitted a paper on PCR to Nature, which the journal declined to publish with the advice that it might better be published in a more specialized journal. Cetus would soon “go upside down,” Mullis notes.
“They didn’t understand what they had, and licensed it to Kodak along with all their drug products,” he says.
By the time the two-year license period elapsed, Cetus apparently realized what it had, and decided to hang on to PCR. Finally, says Mullis, “Hoffman LaRoche bought it for approximately $1 billion.”
Although judging by his website, Mullis seems to have prospered over the intervening years, his share of the spoils was a single $10,000 bonus—and a Nobel Prize.
In the beginning
Subsequent to Mullis’ solitary burst of lightning intuition, PCR became much more of a team sport. Whether Mullis has the price tag right or not, in 1991, Roche did indeed buy the rights to PCR from Cetus and began investing in refining the science for use in molecular diagnostics to detect diseases and for life-science research.
In 1991, the same year that Roche acquired the rights to PCR, reverse-transcription PCR was developed using a single enzyme that withstands heat and can make a DNA strand from RNA, facilitating diagnostic tests for RNA viruses. This was to prove helpful in identifying, studying and better understanding retroviruses and infections like HIV that have RNA instead of DNA in their genomes.
The reverse-transcription PCR process is completed in two steps: the reverse-transcription reaction, followed by PCR amplification. In step one, using a reverse transcriptase in a tube with the RNA sample, RNA is reverse transcribed into a “complementary” DNA (cDNA) strand. This matches the RNA nucleotides with their corresponding DNA nucleotides, creating a DNA sequence. In the second step, using the cDNA, the three step PCR process copies the DNA during multiple cycles of DNA amplification. From there, the DNA may be used in diagnostics and monitoring tests.
In 1991, Applied Biosystems made quantitative PCR (qPCR) commercially viable, easy to use and reliable. The company’s initial concept of TaqMan probes was reported by a team at Cetus, and the technology was subsequently developed by scientists at Applied Biosystems. TaqMan probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end.
In 1996, Applied Biosystems launched the first TaqMan assays for endogenous control genes. Since then, a total of more than 8 million TaqMan assays have been commercialized for a wide range of applications, including gene expression, SNP genotyping, CNV, mutation detection and pathogen analysis. A range of new qPCR methodologies, including stem-loop TaqMan MicroRNA Assays, castPCR, COLD-PCR and others, have been developed for accurate quantitation of short, 22-nt RNA molecules and detection of rare somatic mutations.
In particular, Real-Time PCR has become the gold-standard method for accurate quantification and characterization of nucleic acids, in discovery and routine applications ranging from pathogen detection to mutation discovery and in verification of expression profiles generated on microarrays. Also in 1996, Applied Biosystems was the first to overcome the limitations in quantitation associated with PCR with the introduction of its 7700 Real Time PCR instrument.
“I was amazed when I used this machine at Harvard in early 1997. Finally, we are able to accurately quantify the expression level of our genes of interest,” says Dr. Caifu Chen, who currently serves as vice president of R&D at Life Technologies Corp. in Carlsbad, Calif.
Almost all applications of the technology employ a heat-stable DNA polymerase. In the beginning, that was usually Taq polymerase, an enzyme isolated from the bacterium Thermus aquaticus found in Yellowstone Park’s hot springs.
“New England Biolabs (NEB) was the first company to commercialize Taq DNA polymerase,” says the Ipswich, Mass.-based company’s research director, Dr. Bill Jack. “When Cetus wound up with a patent on Taq, we looked at thermal sea vents and produced the first alternative thermostable DNA proofreading DNA polymerase, Vent DNA Polymerase. It was not a bacterium, but an archae, and very different from Taq. It lasted longer at high temperature and has a proofreading function to make a very faithful copy. Later, at a depth of 2,000 meters, we found Deep Vent DNA Polymerase. I was involved in aspects of the initial cloning, but that effort was lead by another scientist here at NEB, Fran Perler. Her work, along with a number of others here, identified the intein and established that it was a protein-splicing event. My lab was involved in overexpression of Vent DNA Polymerase, and did some of the early work to establish the protein splicing characteristics. Other labs were much more directly involved in the intein work, while my lab examined the enzymology of the polymerase.”
Taq is a low-fidelity enzyme, notes Dr. Edita Smergeliene, who is part of the Thermo Fisher Scientific Inc. group that operates out of the Center of Excellence for Molecular Biology in Vilma, Lithuania. In effect, where formerly there might be one error per 1,000 base fragments, with Thermo Fisher Scientific’s most recently introduced Phusion High-Fidelity DNA polymerases, the error rate has been reduced to one in 52,000 bases.
“PCR is still evolving,” Smergeliene observes.
Technology evolves in real time
Roche’s Molecular Diagnostics unit has done much over the ensuing years to help develop PCR and advance the state of the art. In 2003, Roche released the COBAS TaqMan Analyzer that supported three COBAS TaqMan IVD tests: human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) in Europe. These were the first PCR tests that allow for amplification and detection to occur at the same time. The process was termed Real-Time PCR.
Using fluorescent probes, the DNA produced in the PCR amplification reaction may be monitored in real time to detect the presence of the viral pathogen. As the amplified pathogen DNA accumulates at every PCR cycle, the amplified DNA segments “light up,” allowing a quantitative detection of the virus. Because PCR and detection could now be completed simultaneously, not sequentially, the time to result and human interaction with the samples are decreased, lowering the risk of contamination.
“The company offers systems with a variety of throughputs from 32 to 1,536 samples per run, all delivering increased data reproducibility as a result of their high temperature homogeneity,” says Mike Leous, group marketing manager for qPCR, nucleic acid purification and biochemical reagents.
One of the best-established PCR platforms from Roche for life-science and biopharma research is the LightCycler series of real-time PCR instruments, which was first launched more than 15 years ago. A growing number of drug screening laboratories have started performing fully automated high-throughput qPCR assays on Roche’s LightCycler 1536 System to replace traditional cell-based reporter gene assays in screening, allowing the measurement of endogenous gene expression levels. Many other biopharma labs employ the 384-well LightCycler 480 System to validate gene expression measurements obtained by other methods.
Recent developments
The most recent PCR innovation is the introduction of digital PCR (dPCR), which allows researchers to absolutely count DNA molecules in a sample with an unprecedented higher precision than qPCR. Currently available dPCR systems include Life Technologies’ QuantStudio 3D Digital PCR System, Bio-Rad Laboratories’ QX100 droplet digital PCR (ddPCR) system, Fluidigm’s qdPCR 37K IFC system and RainDance Technologies’ RainDrop Digital PCR.
Richard Kurtz, Bio-Rad Laboratories’ gene expression division marketing manager, agrees with Smergeliene about the continuing evolution and growing applications of PCR. About two years ago, the Hercules, Calif.-based company purchased QuantaLife Inc. for $162 million in cash plus potential future milestone payments. It was, says Kurtz, a “big step, but the interest in QuantaLife’s droplet digital PCR system was compelling. Digital PCR soon proved its value by providing researchers with a new tool for the detection of rare mutations, including distinguishing rare sequences in tumors, precise measurement of copy number variation and absolute quantification of gene expression.”
At the time, Norman Schwarz, Bio-Rad’s president and CEO, said, “We are impressed with QuantaLife’s digital PCR technology and believe it will complement Bio-Rad’s existing amplification business. This elegant solution expands the current state-of-the-art methods of qPCR, and we look forward to its adoption in life-science research.”
And indeed, droplet digital PCR has been widely adopted, with “several hundred systems in place,” Kurtz says. To maintain an entrepreneurial spirit, Bio-Rad formed its Digital Biology Centers and has seen about 30 publications rolling out from its customer base. Just weeks ago, the company launched the QX200 Droplet Digital PCR System, a second-generation unit that provides absolute quantification of target DNA or RNA molecules using EvaGreen or TaqMan hydrolysis probes, providing high sensitivity and precision for various applications. Kurtz points out that the predecessor model, the QX100, has itself been used in high-impact research, including the “Mississippi baby” to measure the very, very low level of HIV” that led to the baby being described as “cured” in some reports.
Among the published studies, one of the common uses of the QX100 has been for rare sequence and rare mutation detection, or “needle-in-a-haystack” problems, such as ultra-sensitive measurements of latent proviral reservoirs in HIV eradication efforts; liquid biopsy studies (e.g., monitoring transplant rejection by abundance of graft DNA in the blood or monitoring cancer patient response to therapy through oncogene markers in cell-free DNA); and detection of somatic CNV mosaicism in human skin cells. Additionally, searches are underway for cerebrospinal fluid biomarkers to identify presymptomatic individuals at risk for Alzheimer’s disease, and for miRNA cancer biomarkers in patient serum.
A further area that is showing great benefit from droplet digital PCR is the measurement of copy number variation (CNV), both in revealing the multi-allelic CNV landscape of the human genome and its potential involvement in inherited disease, and for identifying oncogenic CNVs for personalized treatment of cancer patients. It is also commonly used for making absolute gene expression measurements without the need for a standard curve.
Other established and developing uses are for next-generation sequencing (NGS) library quantification and quality control, validation of NGS results (e.g., for CNVs, RNA editing or differential heteroallelic expression) and for phasing of markers through their physical linkage in droplets.
On the market
Today, PCR is an invaluable tool for molecular biology research and is used daily in academic and pharmaceutical laboratories around the world.
Life Technologies’ digital PCR system, the QuantStudio 3D Digital PCR System, utilizes chip-based PCR. The instrument features a simple workflow with minimal hands-on time.
“The QuantStudio 3D Digital PCR System is the latest revolutionary platform from the team that has delivered every market-leading technology in qPCR,” says Chris Linthwaite, head of genetic analysis at Life Technologies. “By making this novel platform accessible to all labs, we are enabling the democratization of digital PCR and helping the scientific community expand the utilization of this technology in disease research and applications beyond.”
Paco Cifuentes, Life Technologies’ product applications director, says one strength of the QuantStudio 3D Digital PCR system is that unlike droplet dPCR, it is a closed system. After the researcher loads a sample into the chip, the PCR runs on a standard thermal cycler, and the QuantStudio 3D reads the results.
“Once the sample is deposited on the chip, the chip is sealed and disposed of at the end of the experiment,” says Cifuentes. “This approach minimizes the chances for contamination.”
Working at the droplet level, Fluidigm Corp.’s contribution to PCR technology’s continued evolution is based on its microfluidic integrated fluidic circuit (IFC).
“Fluidigm’s IFC contains precisely engineered channels, flow control valves and reaction chambers that deliver a new level of reproducibility and throughput to PCR technology,” says Dave Ruff, the company’s principal scientist. “Each PCR microfluidic reaction chamber uses only nanoliter scale volumes of sample and PCR mastermix, significantly reducing cost and increasing precision, reproducibility and throughput for PCR users. Since Fluidigm’s microfluidic integrated fluidic circuits encase a high density of PCR reaction chambers (reaching into many thousands per chip), applications requiring massively parallel individual PCR reactions—such as digital PCR—are a natural fit.”
Fluidigm has developed a series of Dynamic Array integrated fluidic circuits that allow from 48 to 192 samples to be addressed in parallel. Associated with the revolutionary chip technology are Fluidigm’s platform systems that load the IFCs and thermal cycle and read the resultant qPCR signals. For instance, the BioMark HD System provides technological flexibility to produce high-quality qPCR results for DNA, RNA, miRNA and protein quantification. Furthermore, Fluidigm’s microfluidic and PCR technology nicely synergize to simplify and power the frontiers of single-cell analysis.
Ruff notes that “to fully unleash the power and accuracy of digital PCR, many hundreds to thousands of PCR reactions need to be precisely performed in parallel. Perhaps one of the biggest advantages of using Fluidigm technology to conduct digital PCR is the Fluidigm system’s ability to do real-time reads on digital samples. This gives Fluidigm a leg up in the competitive market because it is currently the only company that provides this capability.”
Fluidigm’s future? Ruff sees it this way: “The future holds tremendous excitement for our PCR technology. For example, the single-cell research community is accelerating its pace of discovery and demand for our technology. DeciBio, a market research company, recently projected that single-cell genomics would be one of the fastest-growing areas of life-science research over the next five years with a compound annual growth rate of 39 percent. The C1 Single-cell Auto Prep System provides researchers the freedom to operate flexible experiments that were not possible just one year ago. The C1 System provides a format to seamlessly customize on-chip workflows to yield amplified products for input into qPCR or next-generation sequencing. New chemistry and microfluidic technologies are being developed to quantify multiple classes of biomarkers in individual, single cells. This will further empower systems biology approaches to help connect the dots between genotype, mRNA, miRNA and protein expression. As researchers continue to scale up their single-cell studies into the 1000s and beyond per project, we can envision in the near future 100,000 single-cell samples becoming a practical experimental scale to undertake studies addressing the complex biology in challenges such as cancer, development and immunology, stem cell heterogeneity and neurological disorders.”
Increasing standards and pushing the envelope
Until recently, the standard for measuring DNA changes has been qPCR, which is a comparative technology, where you have to normalize your answers according to known standards, and you have to have multiple replicates in order to accurately confirm your input as well, points out Dr. Michael Samuels, the principal research scientist and scientific liaison at RainDance Technologies in Billerica, Mass.
“There is also a theoretical limit of a twofold change, representing a single PCR cycle threshold,” Samuels notes.
Digital PCR removes the limitations seen in qPCR by using the power of partitions. No longer are interesting genes compared to a known standard, but rather, they are given absolute values. When an assay has been optimized, a single well can provide absolute quantitation in total copies or copies per microliter. By partitioning into droplets, rare events of interest can have the chance to be found, copies can be counted accurately, very small amounts of virus can be detected, cancer may be found by looking for tumor markers in blood—and much more. Researchers are able to multiplex up to 10 genes at the same time by simply using varying concentrations of just two probes in a single well. The partitions remove most of the competition, allowing the target genes to be amplified and visualized on a 2D plot.
Here are applications RainDance lists where dPCR has been successfully demonstrated:
- Rare allele detection in heterogeneous tumors or other genetic-based diseases
- Liquid biopsies of solid tumor burden using peripheral body fluids
- Non-invasive prenatal diagnostics
- Viral load detection
- Gene expression
- Copy-number variation in heterogeneous samples
- Assays with limited sample material, such as single-cell gene expression and FFPE samples
- DNA quality-control tests before sequencing
- Validation of low-frequency mutations identified by sequencing
Digital PCR measurements are performed by dividing the sample and qPCR assay mixture into a very large number of separate small-volume reactions, such that there is either no or one target molecule present in any individual reaction. This is the fundamental concept for making “digital” measurements, RainDrop’s Samuels points out.
When operating in the digital range (where all reactions contain either no or one target molecule), it is possible to multiplex qPCR assays without concern for competition or cross reactivity, as each single-molecule target-containing reaction will proceed with the target binding to its specific primers and probe, whereas no amplification will occur in reactions without targets. Having each single-target molecule in a separate reaction allows both highly abundant and low-abundant targets to be counted in the same experiment without concern for “swamping out” the low-abundant target (since each separate reaction has at most one target, independent of its concentration in the average sample volume).
RainDance Technologies offers the RainDrop dPCR System for digital PCR, utilizing the company’s patented picodroplet technology to generate up to 10 million droplets in a single well. In approximately 30 minutes’ time in eight separate wells, 80 million droplets can be created. This allows researchers to create up to one billion droplets per day, with each droplet encapsulating a single molecule. With a standard detection capability of one mutant in 250,000 wild-type genes and a dynamic range of six logs, the RainDrop dPCR System can accurately quantitate extremely rare alleles, such as circulating tumor biomarkers like IDH1 for glioma patients, or KRAS for colorectal cancer patients.
“Researchers who desire to push the envelope,” Samuels says, “and go for even rarer mutants will find that the RainDrop dPCR System has a lower limit of detection of one mutant in one million.”
“Not only does the RainDrop dPCR System provide unrivaled sensitivity,” Samuels states, “it allows researchers to go beyond the typical multiplexing capabilities of qPCR. Using two probes with varying intensities, researchers are able to get up to a 10-plex assay done in a single sample. Researchers can find multiple SNPs, housekeeping genes and other potential genes of interest in a single well, unleashing true digital potential.”
(Click here to read part two of this special report)
