Research team led by Nagoya University develop a label-free method for detecting DNA amplification in real time based on refractive index changes in diffracted light.
Polymerase chain reaction (PCR) is a simple and ubiquitous method in molecular biology for amplifying DNA segments into millions of copies. This is important not only for basic research, but also in diagnostics, forensics, and medical applications.
Quantitative real-time PCR is a modified version that incorporates fluorescence labeling to cumulatively measure DNA amplification, rather than monitoring it at the end of the process, as in conventional PCR. Real-time PCR therefore enables sensitive quantification of the amount of the initial DNA template. However, current techniques may introduce bias through sequence errors, pipetting inaccuracies, or unequal binding of fluorescent probes (hybridization).
A research team centered on Nagoya University has now developed a novel method of measuring real-time DNA amplification that is label-free, thus avoiding the bias issues associated with other procedures. The research and its outcomes were reported in Scientific Reports.
Existing label-free detection systems rely on surface immobilization of target molecules, which is expensive, laborious, and ineffective over time. This new method also introduces an element of hybridization bias because of complementary probe binding.
The new technique instead detects changes in the intensity of diffracted light from a laser beam passing through miniscule 200 nm (0.0002 mm)-wide nanochannels filled with analytical sample liquids. The 532-nm laser beam is focused by a lens and then diffracted by passing through the nanochannel and detected by a photodiode. Silica substrates were used to make the nanochannels, and the larger the difference between refractive indices of sample liquids and silica, the smaller is the change in diffracted light intensity, and vice versa.
“We used this technique to provide the first label-free detection of human papillomavirus and the bacteria responsible for tuberculosis,” first author Takao Yasui says. “The method is highly sensitive, and allows quantification of a wide range of initial DNA concentrations, from 1 fM to 1 pM (a 1,000-fold range), so is superior to existing fluorescence-based detection systems.”
“Our system also measures DNA amplification at the relatively low temperature of 34°C and without the need for thermal cycles,” coauthor Noritada Kaji says. “Because it has the potential to be constructed as a single chip and can detect sample volumes as small as 1 µl, which is 100–1,000 times less than conventional detectors are capable of, it is particularly suited to development as a miniaturized form of diagnostics and microbe detection.”
The article, “Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating” was published in Scientific Reports at DOI:10.1038/srep31642
This research was supported by the Cabinet Office, Government of Japan and the Japan Society for the Promotion of Science (JSPS) through the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program), Nanotechnology Platform Program (Molecule and Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, the JSPS Grant-in-Aid for JSPS Fellows, the JSPS Institutional Program for Young Researcher Overseas Visits, the JSPS Grant-in-Aid for Young Scientists (B) 25790028, a Grant-in-Aid (No. 15H01599) for Scientific Research on Innovative Areas “Innovative Materials Engineering Based on Biological Diversity” from MEXT, the Asahi Glass Foundation, and PRESTO, Japan Science and Technology Agency (JST).
Koomi Sung | Research SEA
Symbiotic bacteria: from hitchhiker to beetle bodyguard
28.04.2017 | Johannes Gutenberg-Universität Mainz
Nose2Brain – Better Therapy for Multiple Sclerosis
28.04.2017 | Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik IGB
More and more automobile companies are focusing on body parts made of carbon fiber reinforced plastics (CFRP). However, manufacturing and repair costs must be further reduced in order to make CFRP more economical in use. Together with the Volkswagen AG and five other partners in the project HolQueSt 3D, the Laser Zentrum Hannover e.V. (LZH) has developed laser processes for the automatic trimming, drilling and repair of three-dimensional components.
Automated manufacturing processes are the basis for ultimately establishing the series production of CFRP components. In the project HolQueSt 3D, the LZH has...
Reflecting the structure of composites found in nature and the ancient world, researchers at the University of Illinois at Urbana-Champaign have synthesized thin carbon nanotube (CNT) textiles that exhibit both high electrical conductivity and a level of toughness that is about fifty times higher than copper films, currently used in electronics.
"The structural robustness of thin metal films has significant importance for the reliable operation of smart skin and flexible electronics including...
The nearby, giant radio galaxy M87 hosts a supermassive black hole (BH) and is well-known for its bright jet dominating the spectrum over ten orders of magnitude in frequency. Due to its proximity, jet prominence, and the large black hole mass, M87 is the best laboratory for investigating the formation, acceleration, and collimation of relativistic jets. A research team led by Silke Britzen from the Max Planck Institute for Radio Astronomy in Bonn, Germany, has found strong indication for turbulent processes connecting the accretion disk and the jet of that galaxy providing insights into the longstanding problem of the origin of astrophysical jets.
Supermassive black holes form some of the most enigmatic phenomena in astrophysics. Their enormous energy output is supposed to be generated by the...
The probability to find a certain number of photons inside a laser pulse usually corresponds to a classical distribution of independent events, the so-called...
Microprocessors based on atomically thin materials hold the promise of the evolution of traditional processors as well as new applications in the field of flexible electronics. Now, a TU Wien research team led by Thomas Müller has made a breakthrough in this field as part of an ongoing research project.
Two-dimensional materials, or 2D materials for short, are extremely versatile, although – or often more precisely because – they are made up of just one or a...
28.04.2017 | Event News
20.04.2017 | Event News
18.04.2017 | Event News
28.04.2017 | Medical Engineering
28.04.2017 | Earth Sciences
28.04.2017 | Life Sciences