分子生物学
聚合酶链反应
聚合酶
重组酶聚合酶扩增
底漆(化妆品)
聚合酶
底漆延伸
寡核苷酸
单核苷酸多态性
多重连接依赖探针扩增
DNA
环介导等温扩增
基因组DNA
多重位移放大
化学
生物
遗传学
核苷酸
基因
基因型
DNA提取
外显子
有机化学
水热
作者
Mayreli Ortiz,Míriam Jauset-Rubio,David Kodr,Anna Šimonová,Michal Hocek,Ciara K. O’Sullivan
标识
DOI:10.1016/j.bios.2021.113825
摘要
Hypertrophic cardiomyopathies (HCM) are the principal cause of sudden cardiac death in young athletes and it is estimated that 1 in 500 people have HCM. The aim of this work was to develop an electrochemical platform for the detection of HCM-associated SNP in the Myosin Heavy Chain 7 (MYH7) gene, in fingerprick blood samples. The platform exploits isothermal solid-phase primer elongation using recombinase polymerase amplification with either individual or a combination of four ferrocene-labelled nucleoside triphosphates. Four thiolated reverse primers containing a variable base at their 3' end were immobilised on individual gold electrodes of an array. Following hybridisation with target DNA, solid phase recombinase polymerase amplification was carried out and primer elongation incorporating the ferrocene labelled oligonucleotides was only detected at one of the electrodes, thus facilitating identification of the SNP under interrogation. The assay was applied to the direct detection of the SNP in fingerprick blood samples from eight different individuals, with the results obtained corroborating with next generation sequencing. The ability to be able to robustly identify the SNP using a 10 μL fingerprick sample, demonstrates that SNP discrimination is achieved using low femtomolar (ca. 8 × 105 copies DNA) levels of DNA.
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