Optimisation of DNA hybridisation and toehold strand displacement from magnetic bead surfaces

Abstract

Here, we present work on the improved hybridisation and release of specially designed pegylated double stranded DNA strands with a pendant toehold. For this forensically relevant DNA namely amelogenin (AMEL) (a sex determination gene) and the human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) short tandem repeat (STR) were employed. Magnetic beads functionalised with oligonucleotide capture probes with complementarity to the dsDNA PCR product toehold were fabricated. Characterisation of the beads was achieved through dynamic light (DLS) scattering and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy showing successful attachment of the oligonucleotides. The time frame of hybridisation between the dsPCR product toehold and the oligonucleotide modified beads was decreased from 6 h to 10 min by pre-incubation of the oligonucleotide functionalised magnetic beads in 2% aqueous sodium dodecyl sulphate (SDS) solution. Fluorescent microscopy was used to determine the time of toehold mediated strand displacement of the captured dsPCR product from the magnetic bead. Initially, displacement was achieved within 6 h. This was subsequently reduced to 3 h by the addition of a rate accelerator, PEG6000 at 10% v/v.