Branched-chain polyamine beads for sensitive recovery and detection of low-copy DNA in aqueous solutions

Abstract

N⁴-bis(aminopropyl)spermidine (BCPA), a branched-chain polyamine, is uniquely found in hyperthermophiles that thrive above 80°C. Compared to linear polyamines such as spermidine and spermine, BCPA induces DNA compaction at significantly lower concentrations and precipitates DNA more efficiently. To harness these properties, BCPA was immobilized onto N-hydroxysuccinimide (NHS)-activated magnetic microbeads, and its DNA recovery efficiency was evaluated using Salmon sperm DNA. BCPA-conjugated beads exhibited superior DNA-binding capacity compared to spermidine-conjugated and conventional silica beads, with bound DNA remaining unreleased upon treatment with 0.5 % sodium dodecyl sulfate (SDS), 2 mM ATP, or 2 mM phosphate (Pi) at pH 8.8. However, efficient DNA release was achieved with 2 mM pyrophosphate (PPi) at pH 8.0 or 1 mM PPi at pH 10.3. This property enables direct DNA amplification without a separate release step, as dNTPs used in PCR generate PPi as a byproduct, facilitating DNA detachment. To assess the beads' applicability for low-copy DNA detection, plasmid DNA containing the Ureaplasma parvum 16S rRNA region was prepared in saline at varying concentrations. BCPA-conjugated beads successfully recovered and directly amplified as few as 10³ copies of plasmid DNA from a 10-mL saline solution, whereas the same amount remained undetectable using conventional magnetic beads ethanol precipitation. These findings demonstrate the potential of BCPA-conjugated beads for efficient DNA capture and direct amplification, with promising applications in clinical diagnostics and environmental DNA monitoring.