Articles producció científica> Enginyeria Química

Capture, detection and purification of dsDNA amplicons using a DNA binding protein on magnetic beads

  • Datos identificativos

    Identificador: imarina:9282888
    Autores:
    Ruhela, AnkurSkouridou, VassoMasip, Lluis
    Resumen:
    Magnetic separation has been widely exploited for capture and detection of nucleic acids, including amplicons. Streptavidin-magnetic beads (SA-MB) are typically employed for this purpose, as well as in biosensing applications. However, remaining biotinylated primer in the amplification reaction can compete with labeled amplicon for binding to the beads. Also, the harsh conditions needed for elution of bound amplicons restrict their use for purification purposes. Herein we show that a sequence-specific DNA binding protein immobilized on magnetic beads can serve as an alternative to SA-MB for these applications. This is enabled by the high binding affinity of scCro DNA binding protein for its specific sequence and its ability to bind dsDNA but not ssDNA. This specific sequence is easily incorporated in the amplicon during amplification with an extended primer. The scCro-MB exhibited higher amplicon binding capacity and detection sensitivity compared to SA-MB when both synthetic and genomic DNA were used as templates for PCR. This resulted not only from increased protein load on the beads but also from minimized interference of excess labeled primer remaining in the unpurified amplification reactions. Finally, a proof-of-concept was provided for the use of the scCro-MB for PCR amplicon purification under mild elution conditions using salt.
  • Otros:

    Autor según el artículo: Ruhela, Ankur; Skouridou, Vasso; Masip, Lluis
    Departamento: Enginyeria Química
    Autor/es de la URV: Masip Vernis, Lluis / Ruhela, Ankur / Skouridou, Vasoula
    Palabras clave: Streptavidin Sccro dsdna binding protein Pcr amplicon detection Pcr Magnetic phenomena Magnetic beads Dsdna amplicon purification Dna-binding proteins Dna, single-stranded Dna sequence-specific binding Dna pcr amplicon detection magnetic beads dsdna amplicon purification dna sequence -specific binding
    Resumen: Magnetic separation has been widely exploited for capture and detection of nucleic acids, including amplicons. Streptavidin-magnetic beads (SA-MB) are typically employed for this purpose, as well as in biosensing applications. However, remaining biotinylated primer in the amplification reaction can compete with labeled amplicon for binding to the beads. Also, the harsh conditions needed for elution of bound amplicons restrict their use for purification purposes. Herein we show that a sequence-specific DNA binding protein immobilized on magnetic beads can serve as an alternative to SA-MB for these applications. This is enabled by the high binding affinity of scCro DNA binding protein for its specific sequence and its ability to bind dsDNA but not ssDNA. This specific sequence is easily incorporated in the amplicon during amplification with an extended primer. The scCro-MB exhibited higher amplicon binding capacity and detection sensitivity compared to SA-MB when both synthetic and genomic DNA were used as templates for PCR. This resulted not only from increased protein load on the beads but also from minimized interference of excess labeled primer remaining in the unpurified amplification reactions. Finally, a proof-of-concept was provided for the use of the scCro-MB for PCR amplicon purification under mild elution conditions using salt.
    Áreas temáticas: Química Odontología Molecular biology Medicina ii Medicina i Materiais Interdisciplinar Geociências General medicine Farmacia Engenharias iv Engenharias iii Engenharias ii Educação física Ciências biológicas iii Ciências biológicas ii Ciências biológicas i Ciências ambientais Chemistry, analytical Cell biology Biotecnología Biophysics Biochemistry & molecular biology Biochemistry Biochemical research methods Astronomia / física
    Acceso a la licencia de uso: https://creativecommons.org/licenses/by/3.0/es/
    Direcció de correo del autor: vasoula.skouridou@urv.cat ankur.ruhela@estudiants.urv.cat ankur.ruhela@estudiants.urv.cat lluis.masip@urv.cat
    Identificador del autor: 0000-0002-9712-5429 0000-0001-9407-5844 0000-0001-9407-5844 0000-0002-3938-4633
    Fecha de alta del registro: 2024-10-12
    Versión del articulo depositado: info:eu-repo/semantics/publishedVersion
    Enlace a la fuente original: https://www.sciencedirect.com/science/article/pii/S0003269722003839
    URL Documento de licencia: https://repositori.urv.cat/ca/proteccio-de-dades/
    Referencia al articulo segun fuente origial: Analytical Biochemistry. 658 114923-
    Referencia de l'ítem segons les normes APA: Ruhela, Ankur; Skouridou, Vasso; Masip, Lluis (2022). Capture, detection and purification of dsDNA amplicons using a DNA binding protein on magnetic beads. Analytical Biochemistry, 658(), 114923-. DOI: 10.1016/j.ab.2022.114923
    DOI del artículo: 10.1016/j.ab.2022.114923
    Entidad: Universitat Rovira i Virgili
    Año de publicación de la revista: 2022
    Tipo de publicación: Journal Publications
  • Palabras clave:

    Biochemical Research Methods,Biochemistry,Biochemistry & Molecular Biology,Biophysics,Cell Biology,Chemistry, Analytical,Molecular Biology
    Streptavidin
    Sccro dsdna binding protein
    Pcr amplicon detection
    Pcr
    Magnetic phenomena
    Magnetic beads
    Dsdna amplicon purification
    Dna-binding proteins
    Dna, single-stranded
    Dna sequence-specific binding
    Dna
    pcr amplicon detection
    magnetic beads
    dsdna amplicon purification
    dna sequence -specific binding
    Química
    Odontología
    Molecular biology
    Medicina ii
    Medicina i
    Materiais
    Interdisciplinar
    Geociências
    General medicine
    Farmacia
    Engenharias iv
    Engenharias iii
    Engenharias ii
    Educação física
    Ciências biológicas iii
    Ciências biológicas ii
    Ciências biológicas i
    Ciências ambientais
    Chemistry, analytical
    Cell biology
    Biotecnología
    Biophysics
    Biochemistry & molecular biology
    Biochemistry
    Biochemical research methods
    Astronomia / física
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