Ultrasensitive hybridization capture of short tuberculosis cell-free DNA from urine v2

• DOI: 10.17504/protocols.io.kqdg35bx7v25/v2
• Publication Date: 2024-08-07T16:19:12Z
• Authors: Amy Oreskovic; Barry Lutz

Overview: This protocol describes a method for highly sensitive sequence-specific purification of short tuberculosis (TB) urine cell-free DNA (cfDNA) from large-volume (10 mL) samples. Biotinylated oligonucleotide capture probes complementary to the target interest are immobilized on streptavidin-coated magnetic beads and used capture, concentrate, purify via hybridization. improves upon analytical performance both existing silica-based extraction methods cfDNA previous hybridization protocols, meets several previously unmet design criteria cfDNA: (1) high recovery fragments, (2) large sample input volume, (3) <1 copy/mL sensitivity. There two key innovations that contribute robustness unprecedented sensitivity this method: dual biotinylated probes, which increase compared single by moderating probe density bead surface (a variable affecting efficiency surface-based hybridization), improving thermostability bead-probe linkage, eliminating interference endogenous biotin in urine, two-probe system each region, enables strands double-stranded DNA. We designed short, dilute TB fragments but anticipate it will be versatile may useful other applications types requiring efficient volumes. Design primer sequences new targets is straightforward (see "Materials" tab). Expected performance: Near 100% (95% CI: 82.6 - 117.6%) synthetic ssDNA or dsDNA spiked into 10 mL samples, verified across concentrations at least 1 – 10,000 copies/mL fragment lengths 25 150 bp Limit detection ≤5 copies (0.5 copies/mL) Enables amplification PCR well (500X concentration factor) Tolerant variations composition, including pH (5 8), salt (0 500 mM), non-target µg) Graphic abstract: (A) Schematic design. (B) Overview workflow.