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

• Published in: Analytical biochemistry 2022-12, Vol.658, p.114923-114923, Article 114923
• Main Authors: Ruhela, Ankur, Skouridou, Vasso, Masip, Lluis
• Format: Article
• Language: English
• Subjects: DNA sequence-specific binding; dsDNA amplicon purification; Magnetic beads; PCR amplicon detection; scCro dsDNA binding protein
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2022.114923

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. [Display omitted] •scCro DNA binding protein-magnetic beads are used for PCR amplicon capture and detection.•scCro-magnetic beads are also used for amplicon purification using mild elution conditions.•Amplicons are tagged with the specific DNA binding sequence of scCro using an extended primer.•The scCro-magnetic beads are proposed as alternatives to streptavidin-magnetic beads.•Excess primers do not affect amplicon binding on the beads since scCro binds only dsDNA.