Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma.
Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 μL of CLL blood and 5 μL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard).
Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
Avery
A
Sonnenberg
Jennifer Y
JY
Marciniak
Elaine A
EA
Skowronski
Sareh
S
Manouchehri
Laura
L
Rassenti
Emanuela M
EM
Ghia
George F
GF
Widhopf
Thomas J
TJ
Kipps
Michael J
MJ
Heller
D014408:Biomarkers, Tumor / Q000097:blood* / Q000302:isolation & purification; D001774:Blood Chemical Analysis / Q000379:methods*; D016022:Case-Control Studies; D004273:DNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
Biomarkers; Cancer; Chronic lymphocytic leukemia (CLL); Circulating cell-free (ccf)-DNA; Dielectrophoresis