Articles producció científica> Enginyeria Química

Solid-Phase Primer Elongation Using Biotinylated dNTPs for the Detection of a Single Nucleotide Polymorphism from a Fingerprick Blood Sample

  • Dades identificatives

    Identificador: imarina:9232642
    Autors:
    Jauset-Rubio, MiriamOrtiz, MayreliO'Sullivan, Ciara K
    Resum:
    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.
  • Altres:

    Autor segons l'article: Jauset-Rubio, Miriam; Ortiz, Mayreli; O'Sullivan, Ciara K
    Departament: Enginyeria Química
    Autor/s de la URV: Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli
    Paraules clau: 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
    Resum: 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.
    Àrees temàtiques: 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
    Accès a la llicència d'ús: https://creativecommons.org/licenses/by/3.0/es/
    Adreça de correu electrònic de l'autor: mayreli.ortiz@urv.cat miriam.jauset@urv.cat mayreli.ortiz@urv.cat
    Identificador de l'autor: 0000-0002-9423-0055 0000-0002-9943-6132 0000-0002-9423-0055
    Data d'alta del registre: 2024-10-26
    Versió de l'article dipositat: info:eu-repo/semantics/publishedVersion
    Enllaç font original: https://pubs.acs.org/doi/10.1021/acs.analchem.1c03419
    URL Document de llicència: https://repositori.urv.cat/ca/proteccio-de-dades/
    Referència a l'article segons font original: Analytical Chemistry. 93 (44): 14578-14585
    Referència de l'ítem segons les normes 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
    DOI de l'article: 10.1021/acs.analchem.1c03419
    Entitat: Universitat Rovira i Virgili
    Any de publicació de la revista: 2021
    Tipus de publicació: Journal Publications
  • Paraules clau:

    Analytical Chemistry,Chemistry, Analytical
    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
    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
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