Author, as appears in the article.: Ruhela, Ankur; Skouridou, Vasso; Masip, Lluis
Department: Enginyeria Química
URV's Author/s: Masip Vernis, Lluis / Ruhela, Ankur / Skouridou, Vasoula
Keywords: 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
Abstract: 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.
Thematic Areas: 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
licence for use: https://creativecommons.org/licenses/by/3.0/es/
Author's mail: vasoula.skouridou@urv.cat ankur.ruhela@estudiants.urv.cat ankur.ruhela@estudiants.urv.cat lluis.masip@urv.cat
Author identifier: 0000-0002-9712-5429 0000-0001-9407-5844 0000-0001-9407-5844 0000-0002-3938-4633
Record's date: 2024-10-12
Papper version: info:eu-repo/semantics/publishedVersion
Link to the original source: https://www.sciencedirect.com/science/article/pii/S0003269722003839
Licence document URL: https://repositori.urv.cat/ca/proteccio-de-dades/
Papper original source: Analytical Biochemistry. 658 114923-
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
Article's DOI: 10.1016/j.ab.2022.114923
Entity: Universitat Rovira i Virgili
Journal publication year: 2022
Publication Type: Journal Publications