4-Color Discrimination of Multi-Allele Single Nucleotide Polymorphisms on the Absolute Q

Background

Single nucleotide variants (SNVs) or single nucleotide polymorphisms (SNPs), have been implicated in many diseases. The detection, quantification, and discrimination of SNPs has a myriad of relevant applications in precision medicine. Furthermore, multiplexing a reaction to identify more than one allele target per reaction maximizes the amount of data obtained from a single sample. With high specificity and absolute quantification capabilities, digital PCR (dPCR) offers a technical advantage over many SNP detection or genotyping methods.

The Absolute Q is a fully integrated 4-color digital PCR platform that automates all steps of a typical dPCR reaction including partitioning, thermal cycling, and data acquisition. The microfluidic array partitioning (MAP) plate provides routine and consistent generation of 20,000 identically sized partitions, dispersing over 95 percent of sample across each dPCR reaction, every time. Unlike many available digital PCR systems, the workflow is identical to qPCR, and generates digital PCR results in as little as 90 minutes.

To demonstrate 4-color optical multiplexing for single nucleotide difference discrimination, a 4-plex assay was designed in collaboration with Integrated DNA Technologies for a set of alleles in the CYP2C19 gene (rs12248560). The cytochrome P450 enzyme mediates the primary metabolism of many drugs. Polymorphisms in this gene alter metabolism of certain drug compounds. The polymorphism rs12248560, an ultra-fast metabolism phenotype, has been linked to more favorable outcomes for breast cancer patients receiving the drug tamoxifen.1

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1. Schroth W; Antoniadou L; Fritz P; Schwab M; Muerdter T; Zanger UM; Simon W; Eichelbaum M; Brauch H; “Breast Cancer Treatment Outcome with Adjuvant Tamoxifen Relative to Patient CYP2D6 and CYP2C19 Genotypes.” Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/18024866/.

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