Identification of Mutations in Chronic Lymphocytic Leukemia using ccf-DNA Isolated by Dielectrophoresis
- Author(s): Manouchehri, Sareh
- Advisor(s): Heller, Michael J
- et al.
Circulating cell-free (ccf) DNA has become an important biomarker for the early detection, monitoring, and treatment of cancers. However, the current gold standard methods of isolating ccf-DNA from blood or plasma are labor intensive and time consuming. The complex sample processing procedures may cause degradation or loss of ccf-DNA, and thus greatly inhibit the use of ccf-DNA as a target biomarker for point of care (POC) diagnostics. Therefore, it is essential to develop a rapid and inexpensive ccf-DNA extraction method in order to use ccf-DNA as a biomarker for clinical applications.
The work described in this thesis demonstrates a dielectrophoretic- (DEP) based method for the rapid isolation of ccf-DNA from undiluted whole blood and plasma samples collected from chronic lymphocytic leukemia (CLL) patients. It is demonstrated that DEP can recover ccf-DNA from 25 µL of blood and plasma in less than 15 minutes. To investigate the potential of DEP for use in clinical applications, the dielectrophoreticly recovered ccf-DNA from blood and plasma of CLL patients was amplified using polymerase chain reaction (PCR) and sequenced for CLL specific mutations. The results of the genetic analysis were found to be comparable to sequencing results obtained from ccf-DNA isolated by conventional golden standard methods as well as the DNA extracted directly from B-cells. The ability of DEP-based technology to rapidly isolate ccf-DNA from small volumes of unprocessed blood shows the potential to accelerate sample processing and enable the use of ccf-DNA as a specific biomarker target for point of care diagnostics.