Author, as appears in the article.: Jauset-Rubio, Miriam; Ortiz, Mayreli; O'Sullivan, Ciara K
Department: Enginyeria Química
URV's Author/s: Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli
Keywords: Triphosphate Solid-phase Solid phasis Single nucleotide polymorphisms Single nucleotide polymorphism sites Recombinases Recombinase polymerase amplification Proteins Primer extension Polymorphism Optical detection Nucleotides Nucleic-acid amplification Mycobacterium-tuberculosis Mediated isothermal amplification Hypertrophic cardiomyopathy Gold nanoparticles Enzymatic-synthesis Electrochemical detection Dna Combination Blood samples Blood Biosensor
Abstract: Isothermal recombinase polymerase amplification-based solid-phase primer extension is used for the optical detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample. The assay exploits four thiolated primers which have the same sequences with the exception of the 3'-terminal base. Target DNA containing the SNP site hybridizes to all four of the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under interrogation. Biotinylated deoxynucleotide triphosphates are used in the primer extension, allowing postextension addition of streptavidin-poly-horseradish peroxidase to bind to the incorporated biotinylated dNTPs. The signal generated following substrate addition can then be measured optically. The percentage of biotinylated dNTPs and the duration of primer extension is optimized and the system applied to the identification of a SNP in a fingerprick blood sample. A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 degrees C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The SNP in the fingerprick sample was identified and its identity corroborated using ion torrent next generation sequencing. Ongoing work is focused on extension to the multiplexed detection of SNPs in fingerprick and other biological samples.
Thematic Areas: Química Medicina ii Medicina i Materiais Interdisciplinar Geociências General medicine Farmacia Ensino Engenharias iv Engenharias iii Engenharias ii Enfermagem Ciências biológicas iii Ciências biológicas ii Ciências biológicas i Ciências ambientais Ciências agrárias i Ciência de alimentos Ciência da computação Chemistry, analytical Biotecnología Biodiversidade Astronomia / física Analytical chemistry
licence for use: https://creativecommons.org/licenses/by/3.0/es/
Author's mail: mayreli.ortiz@urv.cat miriam.jauset@urv.cat mayreli.ortiz@urv.cat
Author identifier: 0000-0002-9423-0055 0000-0002-9943-6132 0000-0002-9423-0055
Record's date: 2024-10-26
Papper version: info:eu-repo/semantics/publishedVersion
Link to the original source: https://pubs.acs.org/doi/10.1021/acs.analchem.1c03419
Licence document URL: https://repositori.urv.cat/ca/proteccio-de-dades/
Papper original source: Analytical Chemistry. 93 (44): 14578-14585
APA: Jauset-Rubio, Miriam; Ortiz, Mayreli; O'Sullivan, Ciara K (2021). Solid-Phase Primer Elongation Using Biotinylated dNTPs for the Detection of a Single Nucleotide Polymorphism from a Fingerprick Blood Sample. Analytical Chemistry, 93(44), 14578-14585. DOI: 10.1021/acs.analchem.1c03419
Article's DOI: 10.1021/acs.analchem.1c03419
Entity: Universitat Rovira i Virgili
Journal publication year: 2021
Publication Type: Journal Publications