Abstract

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

Highlights

  • Urine cell-free DNA is generated from transrenal excretion of circulating cfDNA, as well as from local degradation of cells shed along the urogenital tract [1]

  • We report full characterization of the analytical performance of our hybridization capture method and describe two key innovations that contribute to its unprecedented sensitivity: dual biotinylated capture probes and a two-probe system for recovery of both strands of double-stranded DNA

  • Dual biotinylated capture probes complementary to the target of interest are immobilized on streptavidin-coated magnetic beads prior to hybridization

Read more

Summary

Introduction

Urine cell-free DNA (cfDNA) is generated from transrenal excretion of circulating cfDNA, as well as from local degradation of cells shed along the urogenital tract [1]. It is a versatile noninvasive biomarker with applications in cancer detection [1,2,3], infectious disease diagnosis. Ultrasensitive hybridization capture of short urine cell-free DNA from the National Science Foundation Graduate Research Fellowship Program (nsfgrfp.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Objectives
Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call