Genetic Engineering & Biotechnology News

MAY15 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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12 | MAY 15, 2018 | | Genetic Engineering & Biotechnology News That is, it can indicate only if a given DNA sequence is present or absent. But wait: What about real-time PCR, which is also known as quantitative PCR (qPCR)? True to its name, qPCR is indeed quantitative, but only if standard curves are used to convert abundance data—specifically, data about the rate at which abundance increases—into ab- solute concentrations. Granted, qPCR works just fine for many applications. After all, qPCR technology is mature, and qPCR systems are capable of high-throughput operation. Yet qPCR tech- nique may falter in applications requiring extreme accuracy and sensitivity. In such applications, another PCR technique, digital PCR (dPCR), shines. With dPCR, extreme partitioning of samples into arrayed reaction chambers, usually droplets or microwells, ensures that any given chamber contains just one copy of the target sequence, or none. (Yes, the random nature of target distri- bution means that multiple targets may lurk in a single cham- ber, but statistical calculations can take such aberrations into account, never more so than when the number of partitions is large.) Because of its all-or-nothing (1 or 0) approach, dPCR may dispense with standard curves and their compli- cations, and the count of positive signals is easily converted into an absolute number. This relatively straightforward approach delivers a high degree of accuracy and is suitable for challenging applications such as the detection of copy number variations (CNVs) and rare alleles, the analysis of nucleic acid scraps extracted from liquid biopsies, and the identification of elusive pathogens. Recently, dPCR has been coming into its own. Back in the early 1990s, it was still a beguiling curiosity called "limiting dilution PCR" or "single-molecule PCR." It was indepen- dently pursued by laboratories across unrelated disciplines. Then, in 1999, it was dubbed "digital PCR" by Johns Hop- kins researchers who developed a version of dPCR that en- abled them to detect and quantify colon cancer mutations. This version of dPCR was laborious and hard to scale, but improved versions kept coming out, such that the term "digi- tal PCR," little cited as late as 2007, became commonplace— not just in the engineering literature, but in the biomedical literature, too (Morley, 2014, Biomol. Detect. Quantif.) Now dPCR is demonstrating its strengths in various ap- plications. A sampling is presented in this article, which sum- marizes some of the most interesting developments cited at a recent BIO4 Summit, which took place in London and fo- cused on qPCR and dPCR. Technological Improvements According to Erin Zhang, Ph.D., product manager at JN Medsys, this company has developed a dPCR system that has a level of detection down to one copy of DNA per genome. The system, Clarity™ dPCR, can be used to detect DNA, cDNA, or RNA and accomplish rare mutation analysis, de- tect CNVs, and quantify viruses and bacteria. Nonclinical applications include food testing for contamination. "Although the system is still being used primarily as a re- search tool," Dr. Zhang said, "we see strong clinical utilities of dPCR. Plans are underway to obtain the required regula- tory approvals to bring our product into key IVD [in vitro diagnostic] markets." JN Medsys' system uses a "chip in a tube" or tube-strip format that combines the stability of existing droplet-based systems with the speed and workflow advantages of chip- based dPCR systems. The system can complete 96 reactions in less than four hours. The next generation of the Clarity™ Plus system can generate over 40,000 partitions per reaction, allowing multiplexed detection of four to six targets per re- action or 384 to 576 different targets in each dPCR experi- ment. The closed system environment reduces contamina- tion and sample loss and increases the stability of partitions. "With these improvements," Dr. Zhang asserted, "the new system pushes the limits of dPCR analysis by providing better dynamic range and accuracy, and possibly the highest detection throughput." Bio-Rad Laboratories' Droplet Digital™ PCR (ddPCR) system partitions the test sample into 20,000 droplets; after amplification, the droplets containing the target sequence are detected using fluorescence. Like other dPCR technologies, ddPCR dispenses with normalization-to-control methods such as the use of reference samples or standard curves, giv- ing it an advantage over qPCR. "One of the limitations of dPCR is that because it is a PCR technology, it is limited to a handful of targets that can be detected at a time; it's not as broad profiling as NGS," noted Viresh Patel, Ph.D., global marketing director, digital biology group, Bio-Rad. "If you have a limited set of targets, dPCR has advantages over sequencing and NGS—the turn- around time is day or two at most." Dr. Patel said that although dPCR is being applied across applications like those for qPCR and next-generation se- quencing (NGS), dPCR delivers superior performance in mu- tational analysis, detecting single nucleotide polymorphisms (SNPs) and other variants such as deletions, insertions, trans- locations, and fusions in the background of a lot of wild-type or normal DNA. "This ability to find rare variants is where dPCR pro- vides a significant benefit, often log-fold improvements over other methodologies in this area while maintaining cost ef- fectiveness and a simple workflow and turn-around time," Dr. Patel insisted. Other applications of dPCR are detection of pathogens such as viruses or bacteria in the background of complex matrices and wild-type material; quantification of host-cell DNA present in biologic or other pharmaceutical products; and detection of donor-organ DNA in the blood of trans- plant recipients to monitor signs early organ rejection or or- gan failure. Digital PCR is being used to validate and verify successful gene edits in various systems. Bio-Rad provides a self-service OMICS Feature See Digital PCR on page 14 Bio-Rad Laboratories, which supplies digital PCR systems, reagents, and accessories, describes its digital PCR workflow as follows: Left: The DNA sample containing a target sequence is partitioned into droplets, such that each droplet contains one copy or no copies of the target. Middle: The PCR reaction takes place in each droplet. Right: If, in any droplet, the target sequence is amplified, a reporter dye emits a fluorescent signal. Counting the positive droplets gives precise target quantification. With Digital PCR, the Rare Becomes Routine Lynne Lederman, Ph.D. Even standard polymerase chain reaction (PCR) technology should, in principle, boost weak mutant signals so that they may emerge from all the genomic noise emitted by wild-type sequences. Yet standard PCR, for all its sequence-amplifying and signal- boosting powers, is still a qualitative technique.

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