High Sensitivity Detection and Quantitation of DNACopy Number and Single Nucleotide Variants with Single Color DropletDigital PCR

摘要

In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copynumber in the proto-oncogene FLT3 and the commonV600E point mutation in BRAF. Using a series of well-characterizedcontrol samples and cancer cell lines, we confirmed the accuracy ofour method in quantifying mutation percentage and integer value copynumber changes. As another novel feature, our assay was able to detecta mutation comprising less than 1% of an otherwise wild-type sample,as well as copy number changes from cancers even in the context ofsignificant dilution with normal DNA. This flexible and cost-effectivemethod of independent DNA quantification proves to be a robust alternativeto the commercialized TaqMan assay.