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.

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

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.

Dielectrophoretic isolation and detection of cfc-DNA nanoparticulate biomarkers and virus from blood.

Dielectrophoretic (DEP) microarray devices allow important cellular nanoparticulate biomarkers and virus to be rapidly isolated, concentrated, and detected directly from clinical and biological samples. A variety of submicron nanoparticulate entities including cell free circulating (cfc) DNA, mitochondria, and virus can be isolated into DEP high-field areas on microelectrodes, while blood cells and other micron-size entities become isolated into DEP low-field areas between the microelectrodes. The nanoparticulate entities are held in the DEP high-field areas while cells are washed away along with proteins and other small molecules that are not affected by the DEP electric fields.

Dielectrophoretic isolation of DNA and nanoparticles from blood.

The ability to effectively detect disease-related DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for viable diagnostics and therapy monitoring. A DEP method has been developed which allows the rapid isolation, concentration and detection of DNA and nanoparticles directly from human and rat whole blood. Using a microarray device operating at 20 V peak-to-peak and 10 kHz, a wide range of high molecular weight (HMW)-DNA and nanoparticles were concentrated into high-field regions by positive DEP, while the blood cells were concentrated into the low-field regions by negative DEP.

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood.

In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates.

Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences.

Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.

Origins of extrinsic variability in eukaryotic gene expression.

Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes simultaneously, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modelling with fluorescence data generated from multiple promoter-gene inserts in Saccharomyces cerevisiae to identify two major sources of extrinsic variability.

Description and interpretation of adaptive evolution of Escherichia coli K-12 MG1655 by using a genome-scale in silico metabolic model.

Genome-scale in silico metabolic networks of Escherichia coli have been reconstructed. By using a constraint-based in silico model of a reconstructed network, the range of phenotypes exhibited by E. coli under different growth conditions can be computed, and optimal growth phenotypes can be predicted.