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Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood
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Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood
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Journal Article
Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood
2014
Overview
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.
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.
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.
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.
Publisher
Oxford University Press
Subject
/ Biomarkers, Tumor - genetics
/ Biomarkers, Tumor - isolation & purification
/ Blood
/ Cancer
/ DNA
/ DNA, Neoplasm - isolation & purification
/ Electrophoresis, Agar Gel - methods
/ Humans
/ Leukemia
/ Leukemia, Lymphocytic, Chronic, B-Cell - blood
/ Mutation
/ Oligonucleotide Array Sequence Analysis
/ Patients
/ Plasma
