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Goodbye PCR (BioTechniques Newsletter_2013 10)
작성자 이규호 작성일 13-10-04 10:10 조회 3,721
Goodbye PCR
Janelle Weaver (10/02/2013) BioTechniques
PCR has been an invaluable tool in biology, but new PCR-free techniques—from sensors to sequencing—have the promise of revolutionizing clinical care.
Over the past 30 years, PCR has become the workhorse of the modern biology laboratory. But PCR is not the right solution for every problem. For one thing, it can introduce significant bias into datasets. It can also be time-consuming and require skilled technicians, which may not be ideal in circumstances where quick and easy answers are crucial.

The need for fast, simple answers is perhaps most evident in the clinic; the goal of point-of-care tests is to provide doctors and patients with a quick diagnosis. For example, microRNAs—non-coding, single-stranded RNAs that regulate cell growth, differentiation, and apoptosis—have the potential to become promising cancer biomarkers for point-of-care tests. But because of their short length and low abundance, researchers have struggled to detect them. Up to now, microRNA detection methods have required amplification and are not highly sensitive and selective, portable, or low in cost. All of these factors make them unsuitable for point-of-care testing.

To address this need, Chunhai Fan, a physical biologist at the Chinese Academy of Sciences, and his team have developed an ultrasensitive electrochemical microRNA biosensor that does not require PCR amplification. As described last November in 'Scientific Reports', the researchers engineered a 3D DNA tetrahedral nanostructure to achieve high single-base discrimination and attomolar detection sensitivity (1). "The detection limit of 10 attomolar is superior to most state-of-the-art DNA sensors," says Fan. "This is an excellent example of how DNA nanotechnology can revolutionize relatively old technologies."

The sensor also has an extremely large dynamic range, covering nine orders of magnitude, which could allow for simultaneous detection of microRNAs that range drastically in concentration. In the study, Fan and his team demonstrated that the device was capable of detecting altered expression levels of microRNAs extracted from patient tumors. Because the sensor uses inexpensive electrodes and portable electrochemical detectors, it may be useful for clinical diagnostics in the future. However, the biosensor is not without limitations. "While the 10 attomolar sensitivity has been the highest reported for DNA sensors, it is still not possible to detect one to 10 copies of DNA or RNA like PCR can," says Fan. "So, we’re trying to employ a more powerful amplification strategy to further increase the sensitivity."
Picking Up on Pathogens
Shana Kelley, an expert in cellular and molecular sensors at the University of Toronto, has recently reported a similar strategy for the rapid detection of bacterial pathogens that cause infectious disease. Bypassing time-consuming culturing and PCR steps, she has developed a cost-effective platform that combines a universal bacterial lysis approach to release RNA and a sensitive nanostructured electrochemical biosensor. As reported in 2011 in 'Analytical Chemistry',the detection platform combines an electrochemical reporter system and nanostructured microelectrodes to detect specific nucleic acid sequences hybridized to probes immobilized on the sensors (2). "Because the large, 3D chip-based electrochemical sensors promote very efficient binding of target molecules, they are very sensitive and do not require that dilute targets undergo enzymatic amplification for detection," says Kelley. When challenged with unpurified lysates of urine samples spiked with bacteria that cause urinary tract infections, the platform detected the microbes with high sensitivity and specificity and provided results within 30 minutes. The detection limit of the platform allows for uncultured samples to be analyzed.

According to the study authors, this is the first PCR-free, chip-based sensing system to provide sample-to-answer sensing of bacterial pathogens at clinically relevant levels. "This study showed that ultrasensitive detectors could be integrated with a very simple type of sample preparation, which could speed analysis and facilitate eventual use of our methods by untrained users," says Kelley. "The technology is most applicable to infectious disease diagnosis, where fast, streamlined testing can make a big difference in getting infections diagnosed accurately and treated effectively."

But this approach "presupposes that one knows what to target beforehand," says biosensors scientist Grace Hwang from The MITRE Corporation. "In the field of emerging infectious disease, you don’t always have the luxury of knowing what you're looking for," she says. "So this presupposition is acceptable for well-established clinical diseases, but it's not likely suitable for new and emerging threats."

One way that infectious diseases can rapidly spread around the globe is through air travel. Hwang and her colleagues have performed computational simulations to investigate the pathogen-detection sensitivity of commercial off-the-shelf biosensors that do not require PCR and thus can provide answers within 20 minutes without any manual preparation steps (3).

In addition, Hwang has developed optical biosensors for bacterial and viral detection that provide rapid answers without the need for skilled technicians, and her goal is to refine the technology to provide real-time, single-particle pathogen detection in hospitals and airplanes while achieving low false-alarm rates. "Basically, we want to eliminate humans from the loop and get around any kind of reagents or PCR techniques," she says. "It doesn’t make sense for flight attendants to be doing lab work in the event of a disease that required identification."
Seeing the Full Picture
Last month, an analogous approach in the sequencing realm was reported in 'BioTechniques' (4). Paul Coupland, a genomics biotechnologist at the Wellcome Trust Sanger Institute, and his collaborators developed a sequencing method using the Pacific Biosciences RS sequencer that does not require researchers to know ahead of time what to look for and does not require a PCR amplification step or library preparation. "The more library preparation that you do, the more you worry about whether you're seeing the full picture—is it truly representative of the original sample?" says Coupland. "The most important aspect of this work is that you're truly sequencing what you really have in the first place."

This approach requires about eight hours, compared with 12 hours typically needed for library preparation. And the reliance on a small amount of DNA—as little as 1 ng instead of 400–500 ng—is particularly useful in clinical settings because DNA extracted from biopsies is often limited in quantity. At the lowest input amount (0.8 ng), the reads gave enough data to call 91.4% of the genome with 93.4% consensus accuracy. With only 3.1 ng of starting material, mapped reads can cover 100% of the bases at 100% consensus accuracy.

Because the method can be performed without prior sequence knowledge and without organism-specific reagents, it's well suited for examining entire microbial communities as well as acute disease and infec­tious outbreak scenarios. Currently, the direct sequencing method is limited to small genomes due to the relatively low number of reads generated, but it could be used in a clinical setting for sequencing plasmids, single-stranded or double-stranded viruses, mitochondrial DNA, and microbial pathogens.

"PCR-free genome sequencing, I think, will become a highly important diagnostic technique in the next few years," says Coupland. "If you increase the quantity of the data and increase the coverage of larger genomes, that would open the technique to far greater-sized organisms, and hopefully, eventually, to whole human genome sequencing," he adds. "That's the absolute end goal of this technique. It may be a dream that never comes true, but it doesn't seem impossible."
1. Wen, Y., H. Pei, Y. Shen, J. Xi, M. Lin, N. Lu, X. Shen, J. Li, and C. Fan. 2012. DNA nanostructure-based interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA. Sci Rep doi: 10.1038/srep00867.
2. Lam, B., Z. Fang, E. H. Sargent, and S. O. Kelley. 2012. Polymerase chain reaction-free, sample-to-answer bacterial detection in 30 minutes with integrated cell lysis. Anal Chem 84(1):21-5.
3. Hwang, G. M., A. A. DiCarlo, and G. C. Lin. 2011. An analysis on the detection of biological contaminants aboard aircraft. PLoS ONE 6(1): e14520. doi:10.1371/journal.pone.0014520.
4. Coupland, P., T. Chandra, M. Quail, W. Reik, and H. Swerdlow. 2012. Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation. Biotechniques 53(6):365-72.