In our 2011 Future Methods article series, BioTechniques presents in-depth interviews with the thought-leaders and methods developers in the fields of cell biology, genomics, proteomics, epigenetics, and microfluidics. These articles will present the perspectives and thoughts of leading researchers, focusing on the technologies that are being developed to advance research efforts. These articles will provide new information and insights, and a glimpse into how methods and techniques might change in the years to come.
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- Dr. Hood is president and co-founder of the Institute for Systems Biology in Seattle, WA.
- Dr. Wittwer is a professor of pathology at the University of Utah.
- Mr. Banerjee is the president and CEO of RainDance Technologies, Inc.
- Ms. Berdine is director of cancer market development at Life Technologies, Inc.
- Dr. Stevens is director of scientific operations in research and development at Life Technologies, Inc.
BioTechniques: What are some of the technical challenges or limitations of current PCR approaches?
Hood: There are two fundamental limitations of the current PCR technology. The first is the difficulty in designing primer pairs with appropriate specificity without significant optimization because of the lack of comprehensive information on sequence and sequence variations for most of the species.
The second limitation is the frontend sample preparation. DNA is the standard template for PCR, so for RNA samples, a reverse-transcription step is usually employed to convert the RNA molecules into DNA first. This can be difficult for some applications, especially those involving clinical samples because of the low concentration and the presence of inhibitors for reverse transcription enzymes.
In addition, the need to use purified nucleic acids can limit the usefulness of PCR in a clinical setting because it increases the chance of contamination, the time needed for sample preparation, and the amount of starting material required.
Wittwer: A lot of people have gone the direction of doing more of a lot of samples at once. One approach to improve this is to look at the different stages of PCR, denaturation, annealing, and extension, and trying to control them better. If you follow this track, you then look at something as basic as temperature control and the speed of amplification.
From a biochemistry point-of-view, the industrial products have done a poor job in terms of controlling temperature and having enough control to do PCR rapidly. The biochemistry in PCR happens very quickly. The lag, the time concern, is because of the instruments that people have become used to.
Berdine: One challenge that people are facing today is being able to assay large numbers of target in an efficient and cost-effective way. Next-generation sequencing experiments are driving a large number of validation experiments, and people simply have too many targets to validate. So, they usually prioritize and only look at a handful.
Banerjee: Three significant limitations are sensitivity, multiplexing and quantitation. Current standards for PCR at best can detect 1% or perhaps 0.1% mutant in the background of wild type molecules. The “holy grail” is a technology platform that can deliver 1-in-a-million sensitivity, absolute quantitation and multiplexing in a digital way.
BioTechniques: What advantages does a high-throughput approach provide users?
Wittwer: If your focus is on doing more reactions, then you don’t necessarily have to focus on quality of the individual reactions. We’d rather do 10 reactions or 100, 1000, 10,000, or 1 million reactions that will give us what we need. It’s the power of numbers that can overcome the potential uncertainty of what you’re actually amplifying and what the quality might be.
Careful single experiments are losing the argument. There’s extraordinary capability in the number of reactions that are being provided by commercial vendors.
Berdine: We recently launched the QuantStudio 12K Flex, a system that’s very easy to use, has a very streamlined workflow, and can cost-effectively and rapidly analyze a large number of targets for things like next-generation sequencing experiments for genomic experiments that people at contract research organizations are doing. That’s where we’d see the biggest need in terms of qPCR these days.
BioTechniques: On the other hand, are there applications that require better sensitivity than current standards?
Hood:Due to its extreme sensitivity, the reliability of PCR results is always a concern, especially for low-concentration targets. Inaccurate results can be caused by non-specific amplification of unintended targets or by contamination from unwanted template. Although modifications, such as employing high-fidelity DNA polymerases, incorporating chemically modified nucleotides in primer sequences, and adjusting reaction conditions can improve the specificity and accuracy of PCR, we still need to pay attention to prevent or at least reduce the chances for contamination and cross-amplification.
Banerjee: Rare genetic events and low frequency alleles are incredibly hard to identity and validate reliably. But there are clear applications in cancer, infectious disease, immunology and inherited disease research and diagnostics which demand higher sensitivity in the 1:10,000 to 1:1,000,000 range. Examples include detecting low frequency variants in heterogeneous tumors, cell-free mutant DNA in peripheral blood, bacterial identification and strain typing, as well as viral load quantitation. All these require highly sensitive techniques unavailable today.
At RainDance, we’re developing a highly sensitive digital PCR (dPCR) solution capable of analyzing 10 million data points per sample with an easy workflow, for applications that cannot be done by traditional qPCR, arrays or next-generation sequencing because of accuracy, specificity and complex genome reassembly issues.
Stevens: In rare mutation analysis of samples like cancer cells, getting the right sensitivity is a challenge. Life Technologies just released TaqMan mutation-detection assays powered by competitive allele-specific TaqMan (CAST)-PCR. This assay is for the detection of rare allele mutation. CAST-PCR can detect as far as .1%, which would detect very rare events.
The other solution would be dPCR, which allows absolute quantitation as opposed to regular qPCR quantitation. dPCR assays lower the numbers of samples that you need. You don’t need any standard curve. You just do the copy number analysis and either the copy’s there or it’s not.
Berdine: Cancer is one huge opportunity for highly sensitive techniques, in terms of both circulating tumor cells as well as looking at rare events in heterogeneous samples. The other place is in AgBio, such as testing genetically modified foods, to make sure that you have your target of interest and that it’s not a contaminant.
Another one would probably be in infectious disease. You need a level of sensitivity that is far lower than current technology in both sequencing and qPCR allow you to get to.
BioTechniques: Will the ability to look at different analytes become more important for future PCR applications?
Banerjee: PCR has historically been limited by scalability and to single and perhaps 2-3 analytes. This introduces a variety of problems: reproducibility, sensitivity, comparability of different wells and samples especially across different experiments, patients or population studies.
RainDance's digital PCR approach uniquely enables a straightforward approach to multiplexing - we've demonstrated a 5-plex, but we think we can go much higher in the future.
Berdine: In regards to multianalyte analysis, people generally think of TaqMan assays as being able to look at DNA or RNA expression levels. Some of novel technology in proteomics is protein proximity ligation assay (PLA). It allows use of TaqMan assay to look at protein expression levels. So, TaqMan probe can look at all three types of analytes.
BioTechniques: What other new techniques will soon be widely used?
Wittwer: A place where things are getting better in terms of quality would be in a PCR-related technique: DNA melting analysis. Melting analysis is often used as replacement for gel analysis because it can be done basically in a single reaction using a fluorescent dye, just like real-time PCR.
Melting analysis morphed into what is being called high-resolution melting (HRM) analysis. That has opened up a lot of simple applications where it’s easier to detect things than it used to be. This area doesn’t necessarily have the extreme throughput that you get with microPCR reactions, but it’s a very convenient technique that any laboratory can do.
Banerjee: RainDance is launching in mid-2012 the most advanced digital PCR solution in the industry. We have uniquely combined absolute digital quantitation of single molecules of DNA or RNA, unparalleled sensitivity of 10 million data points per sample, the first multiplexed solution in digital PCR, all with a very simple and scalable workflow.
Hood: A small device with a disposable microfluidic chip that integrates sample preparation, amplification reaction, and result interpretation is clearly the future for PCR-based applications. This device can be used at home, the doctor’s office, as well as the field for applications from food safety to pathogen detection.
One of the areas that needs some efforts is in the RNA-based applications. Despite some efforts in the area, a reliable and effective protocol to integrate RNA isolation, reverse-transcription, and PCR amplification is still needed. This would facilitate the development of RNA-based applications, such as measuring the concentration of circulating microRNA in plasma for clinical applications.
Berdine: We have an amazing portfolio in microRNA. What we’re seeing is a pretty rapid adoption of these tools for investigations in epigenetics. Epigenetics is an expanding field. We’re seeing customers not only looking at basic research applications but also at clinical implications of epigenetics, developing drugs that target epigenetic marks.
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Correction: A previous version of this article had incorrectly identified Roopom Banerjee and Anna Berdine. The article has been updated to reflect their correct titles.
