PCR in just minutes, including sample preparation
Because the polymerase chain reaction (PCR) can make exponentially more nucleic acids from DNA or RNA extracted from cells and tissues, the technique has revolutionized medicine, forensics and basic science.
Although PCR can be performed in a few minutes on a small chip, the time-consuming nucleic acid extraction steps have been performed off-chip until now. Jürgen Pipper and colleagues at Singapore’s Institute of Bioengineering and Nanotechnology have developed a pocket-size chip that can do the nucleic-acid extraction steps, essentially eliminating sample preparation by a technician.
The researchers said that they will develop this chip into a point-of-care diagnostic device. In the current demonstration, they used it to quantify DNA that they inserted into leukemia cells that were added to blood.
Using a magnet, researchers moved DNA molecules mixed with magnetic nanoparticles around four positions on a chip, like the 3-, 6-, 9- and 12-hour marks on a watch face. Each position is set at temperatures necessary for PCR. Reprinted with permission of Angewandte Chemie International Edition.
Once placed on the chip, the blood mixed with magnetic nanoparticles attached to antibodies for the leukemia cells. A magnet moved the cells through the DNA extraction process. Then the magnet moved the extracted DNA clockwise through four positions on the chip, each set at temperatures necessary for PCR, as detailed in the May 13 issue of Angewandte Chemie International Edition.
A quantitative variation of the reaction known as real-time PCR was performed on-chip with a commonly used fluorophore and quencher system. Because the fluorescence is quenched until each round of PCR begins, quantitation of the amount of genetic material is possible by measuring the fluorescence during each round.
The fluorescence was excited with filtered light from an Exfo Life Sciences broadband illuminator and was measured with an Olympus microscope and a Hamamatsu photomultiplier tube. In their point-of-care device, the researchers will replace these large instruments with a very small photodetector that can discriminate among various wavelengths, they said.
In the current demonstration of their PCR chip, the researchers extracted and quantified the DNA from 30 cells. Each round of PCR took 8 s, and the entire process lasted 17 min.
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