Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates

Identifier:  imarina:9326298

Handle: https://hdl.handle.net/20.500.11797/imarina9326298

Authors:  Ortiz, Mayreli; Jauset-Rubio, Miriam; Trummer, Olivia; Foessl, Ines; Kodr, David; Acero, Josep Lluis; Botero, Mary Luz; Biggs, Phil; Lenartowicz, Daniel; Trajanoska, Katerina; Rivadeneira, Fernando; Hocek, Michal; Obermayer-Pietsch, Barbara; O'Sullivan, Ciara K

Abstract:
Generic platformusing isothermal solid-phase primer elongationfor cost-effective, rapid electrochemical SNP genotyping from a fingerprickblood sample.Osteoporosis is amultifactorial disease influenced by geneticand environmental factors, which contributes to an increased riskof bone fracture, but early diagnosis of this disease cannot be achievedusing current techniques. We describe a generic platform for the targetedelectrochemical genotyping of SNPs identified by genome-wide associationstudies to be associated with a genetic predisposition to osteoporosis.The platform exploits isothermal solid-phase primer elongation withferrocene-labeled nucleoside triphosphates. Thiolated reverse primersdesigned for each SNP were immobilized on individual gold electrodesof an array. These primers are designed to hybridize to the SNP siteat their 3 & PRIME;OH terminal, and primer elongation occurs only wherethere is 100% complementarity, facilitating the identification andheterozygosity of each SNP under interrogation. The platform was appliedto real blood samples, which were thermally lysed and directly usedwithout the need for DNA extraction or purification. The results werevalidated using Taqman SNP genotyping assays and Sanger sequencing.The assay is complete in just 15 min with a total cost of 0.3europer electrode. The platform is completely generic and has immensepotential for deployment at the point of need in an automated devicefor targeted SNP genotyping with the only required end-user interventionbeing sample addition.