PCR* is widely used in genetic testing. Reportedly, a PCR-based method can detect even a single DNA molecule by amplification. The method is widely used in inspections of GMO (genetically modified organism) foods, cancers, and infections. Some severely strict inspections are prohibited from overlooking any of the specific DNA sequences (target genes). Thus, it is important for testing laboratories to implement thorough quality control over the testing equipment, the reagents, and the detection method as a whole.
Some companies and research institutes have delivered reference materials whose DNA types and densities are prescribed, but they are of high densities—the number of DNA molecules is prescribed in mol (one mol is equivalent to 6.02×1023 DNA molecules). For use in low-density tests at an accuracy of 100 molecules or less, the reference material generally must be diluted. Thus, errors accumulate in the number of molecules distributed while the material is repeatedly diluted. By the time the density reaches the low-density region (particularly where the number of molecules is less than ten), there is a big gap between the expected and actual numbers of DNA molecules, with the resulting samples containing more DNA molecules than prescribed, or conversely no DNA molecules at all.
* PCR (polymerase chain reaction):A method that enzymologically increases the DNA molecules that have a specific sequence
To precisely verify genetic tests, devices and reagents, you need a “scale”—a benchmark in which the number of DNA molecules is determined in a single digit. Through joint research*, Ricoh has developed a technology to manufacture a container of a prescribed number of DNA molecules (a reference DNA plate). Because the number of DNA molecules is accurately controlled molecule-by-molecule, the technology will enable stricter quality control of genetic testers, reagents, and genetic testing methods and increase the reliability of genetic tests. The technology will be useful in preventing modified genes in foods and infections from being overlooked.
* Joint partners:National Agriculture and Food Research Organization (NARO) and FASMAC
In the newly developed process for the reference material, cells are genetically modified to have a target sequence inserted in them enabling the number of DNA molecules containing the target sequence to be counted. The genetically modified cells are counted and delivered from a special bioprinting inkjet head to a 96-well plate; this is done so that each well has a prescribed number of cells. In the end, the cell walls are destroyed to extract the DNAs. That way, a reference DNA plate is manufactured with a determined number of DNA molecules in each well. The technology exploits the high-speed droplet delivery of an inkjet head and allows reference materials to be efficiently produced with the number of DNA molecules precisely controlled.
Cells are as large as 10 µm and can cause sedimentation and nozzle clogging, so it is not easy to deliver them from a generic inkjet head stably. Ricoh has developed a special bioprinting inkjet head for delivering cells. The head has a simple construction without a fluid channel, yet it can deliver fluids in small amounts of solution. Ricoh has also developed a new technology to irradiate the delivered droplets with a pulse laser beam in sync with the delivery, and thus observe the fluorescence of the cells and count the cells in the droplets. The technology enables the droplets to be stably injected into the wells while the cells are counted; the number of cells can now be strictly controlled.
A real-time PCR device was used to evaluate the threshold cycle (the output value of the real-time PCR device, indicating how many times of amplification is required for detection) for 1 to 1,000 DNA molecules. Linearity was demonstrated in the region of 100 or fewer DNA molecules, where sufficient analysis was not possible in the past; it was verified that DNA molecules would be correctly detected even when the number was small. We can logically conclude that using the reference DNA plate will enable production of a precision scale for real-time PCR devices.
In pursuit of the possibilities of printing technologies, Ricoh is seeking possibilities on new customer platforms. In the biomedical field, Ricoh leverages its bioprinting technology and creates new value jointly with other institutes and enterprises through open innovation. The aim is to contribute to providing solutions to social issues.