title: Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood.
abstract: BACKGROUND
  Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications.
  
  
  METHODS
  We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced.
  
  
  RESULTS
  PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood.
  
  
  CONCLUSIONS
  Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.
authors.affiliation: Department of Bioengineering.
authors.forename: Avery
authors.initials: A
authors.lastname: Sonnenberg
authors.forename: Jennifer Y
authors.initials: JY
authors.lastname: Marciniak
authors.forename: Laura
authors.initials: L
authors.lastname: Rassenti
authors.forename: Emanuela M
authors.initials: EM
authors.lastname: Ghia
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Skowronski
authors.forename: Sareh
authors.initials: S
authors.lastname: Manouchehri
authors.forename: James
authors.initials: J
authors.lastname: McCanna
authors.forename: George F
authors.initials: GF
authors.lastname: Widhopf
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kipps
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Heller
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm