A high-throughput protocol for isolating cell-free circulating tumor DNA from peripheral blood

Biotechniques. 2019 Feb;66(2):85-92. doi: 10.2144/btn-2018-0148.

Abstract

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

Keywords: cfDNA; ctDNA; library construction; liquid biopsy; next-generation sequencing; target capture.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / blood
  • Biomarkers, Tumor / isolation & purification
  • Cell-Free Nucleic Acids / blood*
  • Cell-Free Nucleic Acids / isolation & purification
  • Circulating Tumor DNA / blood*
  • Circulating Tumor DNA / isolation & purification
  • High-Throughput Nucleotide Sequencing
  • High-Throughput Screening Assays / methods*
  • Humans
  • Mutation
  • Neoplasms / blood*
  • Neoplasms / genetics

Substances

  • Biomarkers, Tumor
  • Cell-Free Nucleic Acids
  • Circulating Tumor DNA