Whether it is for diagnosing an infectious disease, assessing the gene expression of cancer cells, or identifying microbial contaminants in our water supply, quantitative polymerase chain reaction (qPCR) is the go-to technique of many research labs. In essence, qPCR allows for the amplification and quantification of DNA molecules based on the incorporation of a fluorescent probe.
However, to detect the fluorescence of existing probes, highly sensitive—and therefore expensive—equipment is required to obtain a readout of gene expression. To overcome this barrier to widespread use, a research team led by Ichiro Hirao at A*STAR’s Institute of Bioengineering and Nanotechnology (IBN) developed an expanded genetic alphabet system of artificial DNA bases tagged with molecules that fluoresce intensely when in close proximity.
Hirao first came up with the expanded genetic alphabet at RIKEN, Japan, creating two artificial DNA bases—‘Ds’ and ‘Px’—that pair up, the same way base pairing occurs in the natural DNA bases, between G and C, and A and T. “By increasing the number of base components using unnatural bases, we can produce new DNA molecules with increased functionality,” he said.
Expanding on this idea, Hirao and colleagues began modifying their novel genetic bases to incorporate fluorescent molecules. The researchers synthesized a short DNA strand, called a primer, containing ‘Ds’ and another unnatural base which they called ‘s’. Meanwhile, ‘Px’ was attached to a fluorescent probe, Cy3.
Put simply, during the DNA amplification step in qPCR, ‘Ds’ and ‘Px’ pair up, bringing Cy3 close to ‘s’, resulting in the emission of bright fluorescence visible to the naked eye. Using their technique, the group was not only able to detect the target DNA, but also measure the copy number of DNA molecules based on the intensity of the fluorescence, thereby enabling quantitative, visual PCR.
Finally, the researchers demonstrated that their system could successfully distinguish between bacterial antibiotic resistance genes that differed only by a single nucleotide. They noted that quantitative, visual PCR would be particularly useful for field applications, especially in situations where rapid detection, ease of use and specificity are paramount.
“We would like to use this method to develop diagnostic kits for infectious diseases, such as dengue and Zika. For instance, the dengue virus has four serotypes, and the combination of real-time PCR with our method can identify the dengue serotype of patients at health clinics within a short time period,” said Hirao. The group is now working on an improved PCR method that more precisely amplifies the target DNA sequences.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Bioengineering and Nanotechnology (IBN). A collaboration with the Singapore Institute of Manufacturing Technology (SIMTech) is also in the works, with plans to implement visual PCR in microfluidic devices that could pave the way for easy-to-use, miniaturized diagnostic kits.