Genotyping single nucleotide polymorphisms in human genomic DNA with an automated and self-contained PCR cassette

Abstract

Point-of-care devices can lower costs through reduced reagent costs, shifting diagnostics from centralized laboratories to local clinics or hospitals, rapidly informing on the spot medical decision making, and enabling personalized treatment options. We have previously described a self-contained miniaturized device that uses an array of gel-based reaction units that can simultaneously detect multiple biomarkers and/or multiple patients in one PCR cassette and can be stored for up to 7 months. In this article, we document the ability of cassette PCR to detect single nucleotide polymorphisms (SNPs) in human genomic DNA from buccal swabs. Swab processing takes 8 minutes, and PCR is completed in just more than an hour. To demonstrate potential for genotyping, we used allele-specific PCR and melt curve analysis to detect major and minor alleles of two SNPs in the fibroblast growth factor receptor 2 gene (FGFR2) that are linked with breast cancer. After allele-specific PCR, seamless melt curve analysis and the presence or absence of melt peaks from melt curve analysis identifies the FGFR2 SNP genotypes for each patient. The near point-of-care/point-of-need genotyping methods reported here can be applied for detecting and assessing risks of diseases such as cancer and to detect SNPs that alter drug metabolism and hence response to therapy.