A NOVEL DIGITAL COUNTING ASSAY FOR MOLECULAR DIAGNOSTICS

ABSTRACT Aim: In a study of circulating tumor DNA in the blood of cancer patients, digital PCR and next generation sequencing is used when high sensitivity detection is required. However, these methods include a PCR amplification process, and miselongation of the DNA polymerase may cause a false result. Therefore we have developed a technique for detecting a single molecule without the PCR process by using the signal amplification reactions directly from target DNA. This method can identify the difference of a single base within 15 minutes. And, it can be quantified. Methods: A mix of the reaction reagent and target DNA oligo (∼100base) were put in the glass plate which has an enormous number of micro wells. At this time, the volume of one droplet is less 100 fL. After warming at 62 for 15 minutes on a hot plate, the numbers of bright droplets were counted by a fluorescence microscope. Then, the numbers of bright droplets were counted when the concentration of target DNA oligo increases from 300 fM to 300 pM. In addition, we used the invader reagent which has two type of fluorescence to evaluate that this method could identify the difference of single base of DNA. Invade probes were designed. Results: It was confirmed that the counted bright droplets had a relationship with the Poisson distribution. Further, by using mixture target DNA of mutant-type and wild-type. And also, we found that Red emission was obtained from the hole which contains the wild-type DNA, Green emission was obtained from the hole which contains the mutant-type DNA. Conclusions: We have established a method that quantitates and achieves possible identification of single base of DNA within 15 minutes. In addition, this digital counting method has great possibilities of detecting various biomarkers such as DNA, mRNA and miRNA in blood or tissue of cancer patients. Disclosure: All authors have declared no conflicts of interest.