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Stanford Group Develops Single-Color Droplet Digital PCR Assays for CNV, SNP Detection

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Scientists from the Stanford University School of Medicine and the Stanford Genome Technology Center have demonstrated proof of principle for performing high-sensitivity detection and quantification of DNA copy number and SNPs on Bio-Rad's droplet digital PCR platform using a single-color DNA-binding dye.

According to the researchers, their technique is a viable alternative to multi-color TaqMan-based droplet digital PCR assays, which can be relatively cumbersome, inflexible, and expensive. In addition, their assay may prove to be a streamlined method for targeted mutation analysis in samples where DNA is degraded or scant, such as small biopsies and circulating nucleic acids from blood.

The Stanford researchers, led by Hanlee Ji, presented their work in a poster at the American Association for Cancer Research annual meeting held earlier this month in San Diego. The group also published its technique in an Analytical Chemistry paper in January.

Their work builds on previous research from Bio-Rad scientists, who in November published a paper also in Analytical Chemistry demonstrating the compatibility of the company's latest droplet digital PCR system, the QX200, with DNA-binding dyes such as EvaGreen rather than the fluorescent TaqMan hydrolysis probes that were most commonly used for assay development on Bio-Rad's first-generation platform, the QX100.

In that paper, the Bio-Rad team performed a head-to-head comparison of TaqMan- and EvaGreen-based quantification of five genes, showing that the two approaches yielded equivalent results. In addition, they demonstrated proof of principle for two multiplexing methods based on discernible differences in fluorescence amplitude — one caused by creating different-length amplicons for each target, and one achieved by varying input primer concentrations to create different masses of amplified DNA.

In their recent work, Ji and colleagues focused on the first method — varying the amplitude of EvaGreen signal by toying with amplicon length — and took it a step farther, using the method to detect and quantify CNVs across a wide range of targets and sample sources, and to detect SNPs from cancer cell lines and patient samples.

"Compared to highly variable quantitative real-time PCR, ddPCR eliminates relative standards and has the advantage of measuring mutant and wild-type targets within the same well," Ji and colleagues wrote in their AACR abstract. "Commonly, this is achieved with the use of a two-color fluorescent oligonucleotide probe (TaqMan) design, where the mutant is represented by a FAM probe and the wild type by a VIC or HEX probe. However, this approach is cumbersome and requires a significant amount of optimization."

Instead of using two different-colored fluorescent probes, Ji and colleagues' EvaGreen-based design "manipulates the length of the region of interest and control amplicons to distinguish between their fluorescent signals," they wrote. "The dye binds in greater amount to the longer-length target giving a higher fluorescent signal than the mutant target, and both populations are easily quantifiable. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay."

More specifically, Ji and colleagues tested the ability of their method to quantify the copy number amplification of the oncogene FLT3, and found that they were able to accurately quantify an FLT3 copy number change in a tumor sample harboring a 1.5-fold amplification diluted to 20 percent of a normal sample.

In addition, they designed an assay for the BRAF V600E point mutation using the same concept of amplicon length variation to separate droplet populations. This design incorporated a long, non-complementary tail onto the 5' end of the BRAF wild-type primer and a short tail onto the BRAF mutant primer. "The longer amplicon produced higher-amplitude positive droplets than the shorter amplicon; thus, the population of wild-type positive droplets and mutant positive droplets were distinctly clustered and quantified similar to the CNV assay," they wrote in their Analytical Chemistry paper.

They then tested this assay on a series of diploid control DNA, cancer cell lines, and a colorectal cancer patient sample for which next-generation sequencing had verified the presence of the targeted mutation. In all cases, they compared their method to BioRad's commercially available PrimePCR BRAF V600E assay, which uses TaqMan hydrolysis probes, and found that the reported values from the EvaGreen assay were similar. They also found that their assay was able to detect a mutation comprising less than 1 percent of an otherwise wild-type sample.

Finally, the Stanford researchers used their assay to detect an activating mutation in HER2, and were able to distinguish between wild type and two different mutant alleles.

Overall, Ji and colleagues concluded that their method "retains the accuracy found in TaqMan-based droplet digital PCR platforms while eliminating the need for optimization of the probe oligonucleotide. Sampling error is minimized because both the [region of interest] and the reference gene are measured from the same template."

Further, "since a third oligonucleotide is unnecessary in this system, it is possible to use shorter amplicons, which is preferable in the context of degraded DNA," the researchers wrote. "Whereas TaqMan-based assays are limited by the efficiency of the oligonucleotide probe and dependent on the neighboring nucleotide context, our single-color digital PCR strategy is a highly flexible platform that can be used to interrogate a wide range of genetic targets."

The Ji lab is currently determining whether incorporating locked nucleic acids in their assay design will increase its specificity and sensitivity in lower-quality samples.

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