High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR

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 FLT3 and the common V600E point mutation in BRAF. 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.

Figure 1

Schematic of droplet-digital PCR with EvaGreen dye. Droplets are formed pre-PCR by randomly sequestering fragmented template DNA into equal volume partitions. The first population of droplets corresponding with the lowest fluorescent amplitude has only the unamplified background template DNA (gray). The second population represents the droplets containing only the short amplicon template (black). For CNV analysis, this is UC1 (60bp), and for SNV analysis, this is the short-tail amplicon (71bp). The third population represents droplets with only the long amplicon template (red). For CNV analysis, this is the ROI (66bp), and for SNV analysis, this is the long-tail amplicon (104bp). The population with the highest amplitude represents droplets containing both amplified targets.

Figure 2

Correlated increase in amplicon length and EvaGreen fluorescence amplitude. (A) Reverse primers were designed to flank increasing length regions of FLT3 with a common forward primer. All primers were approximately 20 bp. (B) Each column represents individual wells of ∼20,000 droplets with a single set of FLT3 primers. Amplicons >500 bp were allowed to anneal/extend for two minutes instead of one.

Figure 3

Primer optimization for CNV assay. (A) NA18507 normal diploid genomic DNA served as template for simplex PCR amplification of FLT3 (66bp), UC1 (60bp), and a multiplexed reaction for both targets. Amplicons were run on a nondenaturing TBE acrylamide gel. (B) Each column represents a single well of ∼20,000 droplets containing NA18507 template with multiplexed UC1 and FLT3.

Figure 4

Sensitivity testing of EvaGreen CNV assay on control DNA. (A) G6PD copy number measured in four human X-chromosome disorder DNA samples (Coriell). The expected one-to-one line (orange) is based on the copy numbers provided by the Coriell Institute. (B) FLT3 copy number as measured in a serial dilution of NCIH716 colorectal cancer cell line into a normal diploid human control, NA18507 (Coriell). The expected copy number (green) was calculated from the Cancer Cell Line Encyclopedia (Broad) data on copy number derived from microarray analysis.

Figure 5

One-color SNV quantification. (A) Primers are designed with the single nucleotide variant at the 3′ end of the complementary region. Noncomplementary tails of varying lengths are then added to the 5′ end and amplified with a universal reverse primer. (B) 1:4 mixture of MUT/WT BRAF template amplified with mutant primers with the short tail and wild-type primers with the long tail. (C) Swap: 1:4 mixture of MUT/WT BRAF template amplified with wild-type primers with the short tail and mutant primers with the long tail. (D) Serial dilution of mutant BRAF template (LS411N) into wild-type (Human male control). Theoretical % mutant was calculated from TaqMan measured concentrations of mutant and wild-type template. The assay was performed with the EvaGreen primer mix from (B). (E) The red border regions provide a magnified view of three data points on the lower end of the dilution series from (D).