Technology

ddPCR

Polymerase Chain Reaction (PCR) strategies have dramatically changed in recent years from the standard 1^st generation in which target DNA(s) are amplified to endpoint, or saturation, and products are then quantified using gel-electrophoresis and the resulting band intensity. Real-time PCR, considered to be the 2^nd generation PCR technology, measures the fluorescent signal of a target DNA molecule over the course of a PCR reaction. Both of these technologies enable the user to generate information about the relative concentration or quantity of their target of interest. (Hindson et al., 2011)

Digital Droplet PCR can be considered the 3^rd generation of PCR technologies. Using oil-emulsion technology, the user can partition a standard PCR reaction into tens of thousands of individual micro-volume droplet reactions. Each droplet contains individual PCR reaction which is cycled to endpoint much like 1^st generation PCR. The key advantage of digital PCR over the bulk 1^st generation technology is that upon partitioning template DNA is isolated such that many the droplets contain no template DNA and only a handful of droplets contain 1 or fewer DNA molecules. Upon cycling all droplets to endpoint, the user can interrogate individual droplets to identify those which originally contained the DNA target of interest. This enables unparalleled precision down to single molecule resolution and builds

We developed a molecular test that detects several important cancer mutations found in different tumors – this includes colon and bile duct cancer. Our approach enables one to generate tests that are customized for an individual patient.(Wood-Bouwens, Lau, Handy, Lee, & Ji, 2017) The mutations were selected based on mutations present among six cancers from different patients. Using cancer cell lines that one grows within a plastic dish in the laboratory, we determined the performance of these customized molecular tests. In these controlled conditions, the test worked extremely well and was so sensitive that it could detect as low as three DNA molecules with a cancer mutation. Subsequently, we used these customized molecular tests in a clinical study. Namely, we focused on detecting “personal” cancer mutations from the blood of six different patients. Three of these patients had cancer mutations present in their blood. This result generally correlated with the known burden of cancer per imaging studies in each patient.

Single-color single-nucleotide variant mutation detection of single-allele (ie, simplex) and paired-allele genotyping assays. A: Simplified representation of a patient-derived sample assay using ddPCR technology. (1) Spin whole blood to isolate plasma, (2) extract circulating cell-free DNA, and (3–5) single-color ddPCR workflow and output. B: A single-allele genotyping assay is designed with a common forward primer that targets genomic DNA paired with an allele-specific reverse primer, thus targeting one allele per ddPCR. The allele-specific base is the last 3′ base of the primer. A paired-allele genotyping assay, in the context of this article, refers to combining the wild-type allele genotyping assay with the mutation allele genotyping assay in one ddPCR. To facilitate separation of droplets based on fluorescence, a noncomplementary tail is added to one of the allele-specific primers, herein shown as the mutation.

Tracking a patient’s tumor burden is a critically important part of his or her medical management. For monitoring patients’ tumors, there are only a handful of blood tests limited to only several types of cancers. Instead, nearly all cancer patients require whole body imaging, such as CT scans, to determine the presence and growth of their tumor. Given the high cost and complexity of imaging studies, these scans are done at long intervals of months at a time if not longer. In contrast, molecular tests like the one we have developed will enable patients to be monitored at every visit and thus have the potential for quickly monitoring cancer growth and spread. In addition, the test’s rapid turnaround and relatively low cost provide a potential opportunity for universal monitoring of more patients than is currently done.

Hindson, B. J., Ness, K. D., Masquelier, D. A., Belgrader, P., Heredia, N. J., Makarewicz, A. J., . . . Colston, B. W. (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem, 83(22), 8604-8610. doi:10.1021/ac202028g

Wood-Bouwens, C., Lau, B. T., Handy, C. M., Lee, H., & Ji, H. P. (2017). Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA. J Mol Diagn, 19(5), 697-710. doi:10.1016/j.jmoldx.2017.05.003