Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood

Avery Sonnenberg, Jennifer Y. Marciniak, Laura Rassenti, Emanuela M. Ghia, Elaine A. Skowronski, Sareh Manouchehri, James McCanna, George F. Widhopf, Thomas J. Kipps, Michael J. Heller

Research output: Contribution to journalArticle

33 Scopus citations

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 anACelectrokinetic 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.

Original languageEnglish (US)
Pages (from-to)500-509
Number of pages10
JournalClinical chemistry
Volume60
Issue number3
DOIs
StatePublished - Mar 2014

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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    Sonnenberg, A., Marciniak, J. Y., Rassenti, L., Ghia, E. M., Skowronski, E. A., Manouchehri, S., McCanna, J., Widhopf, G. F., Kipps, T. J., & Heller, M. J. (2014). Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood. Clinical chemistry, 60(3), 500-509. https://doi.org/10.1373/clinchem.2013.214874