Fatemeh Mansouri - urmia university of medical sciences

The origins and principles of digital PCR as a new clinical diagnostics based on the ability of absolute quantitation to detect a single molecule of the target genes. This technology is used for detection of DNA, RNA, microRNA and other biomarkers with a limited number of targets and a range of small concentrations. The samples must be diluted and divided into a large number of aliquots so that some aliquots receive one molecule of the target gene. The number of positive aliquots, as performed on thermocycler reflects the abundance of the target genes in the sample and measure flourescence signal after each cycle of PCR. Positive samples have increased fluorescence against negative samples and quanta software measures the number of positive and negative samples per fluorescence. It provides absolute and extremely high resolution for target quantification without the need for a standard curve and makes the correct measurement of very few copies of a target. If the sample is sufficiently dilute, only a few of the aliquots will be positive and each of these positive aliquots can be assumed to have contained only a single target molecule. Digital PCR only detects specific targets DNA or RNA and cannot detect proteins or degraded DNA molecules and adjusted for multiple targets. Although this method is very expensive but is very suited for detection individual biomarkers in cancer with a limited number of targets molecules (DNA, RNA, miRNA) in all biofluid samples, Circulating tumor cells and tumor surrounded by normal cells. Moreover, digital PCR is a robust method for quantifying genome amplification, fetal DNA in maternal blood and copy number variation in formalin fixed paraffin embedded in tumors, cardiovascular disease and diabetes.

Clinical diagnostic, Digital PCR, Biomarker