High Sensitivity Detection and Quantitation of DNA
Copy Number and Single Nucleotide Variants with Single Color Droplet
Digital PCR
- Publication date
- Publisher
Abstract
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 copy
number in the proto-oncogene <i>FLT3</i> and the common
V600E point mutation in <i>BRAF</i>. Using a series of well-characterized
control samples and cancer cell lines, we confirmed the accuracy of
our method in quantifying mutation percentage and integer value copy
number changes. As another novel feature, our assay was able to detect
a mutation comprising less than 1% of an otherwise wild-type sample,
as well as copy number changes from cancers even in the context of
significant dilution with normal DNA. This flexible and cost-effective
method of independent DNA quantification proves to be a robust alternative
to the commercialized TaqMan assay