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.

Advances in Directly Amplifying Nucleic Acids from Complex Samples

“…Rodriguez et al used a similar approach in detecting clinical levels of H1N1 by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [205]. Future developments could focus on direct reactions with swabs that integrate extraction, Typically, elution either at room temperature [51, 176,[182][183][184][185][186] or with heating [43,44, [187][188][189][190][191] is sufficient to generate PCR-amplifiable template from swabs in solution. These methods of dilution or heating can save over thirty minutes of processing time, as with Nihonyanagi et al's heating protocol to release MRSA in CellEaseII (Biocosm Inc., Hyogo, Japan) diluents [169].…”

Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting Escherichia coli in Beef Carcass Swabs

“…With the smaller size, low cost, and rapid testing capabilities, miniaturized lab-on-a-chip devices can change the way food-borne pathogen detection is currently performed [ 8 , 9 ]. We have demonstrated such a device termed “cassette PCR” that is self-contained, simple, disposable, and inexpensive [ 10 , 11 , 12 , 13 , 14 ]. In cassette PCR, PCR is performed in a semi-solid gel, followed by in situ MCA in a capillary reaction unit of ~6 μL volume.…”

Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR

“…These pre-made cassettes can be stored for at least 7 months at 4 °C or − 20 °C [10] and up to 3 years at 4 °C (unpublished results). With this technology, we have previously detected multiple sexually transmitted diseases for multiple patients with raw urine and genital swabs [11] and single nucleotide polymorphisms in breast cancer genes tested using buccal swabs [12]. With a somewhat different architecture for making reaction gels, we also amplified BK virus in whole blood with no purification involved [13].…”