Ortiz Mayreli, Jauset-Rubio Miriam, Trummer Olivia, Foessl Ines, Kodr David, Acero Josep Lluís, Botero Mary Luz, Biggs Phil, Lenartowicz Daniel, Trajanoska Katerina, Rivadeneira Fernando, Hocek Michal, Obermayer-Pietsch Barbara, O'Sullivan Ciara K
INTERFIBIO Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain.
Division of Endocrinology and Diabetology, Department of Internal Medicine, Medical University of Graz, 8036 Graz, Austria.
ACS Cent Sci. 2023 Jul 19;9(8):1591-1602. doi: 10.1021/acscentsci.3c00243. eCollection 2023 Aug 23.
Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.
骨质疏松症是一种受遗传和环境因素影响的多因素疾病，它会增加骨折风险，但目前的技术无法实现对该疾病的早期诊断。我们描述了一个通用平台，用于对全基因组关联研究确定的与骨质疏松症遗传易感性相关的单核苷酸多态性（SNP）进行靶向电化学基因分型。该平台利用二茂铁标记的三磷酸核苷进行等温固相引物延伸。为每个SNP设计的硫醇化反向引物固定在阵列的各个金电极上。这些引物设计为在其3'OH末端与SNP位点杂交，并且引物延伸仅在存在100%互补性的地方发生，便于对每个被检测的SNP进行鉴定和杂合性分析。该平台应用于实际血液样本，这些样本经过热裂解后可直接使用，无需进行DNA提取或纯化。结果通过Taqman SNP基因分型检测和桑格测序进行验证。该检测仅需15分钟即可完成，每个电极的总成本为0.3欧元。该平台完全通用，在需要时部署在自动化设备中用于靶向SNP基因分型具有巨大潜力，最终用户唯一需要进行的干预是添加样本。
