The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.

Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

The separation and subsequent isolation of the metastatic human cervical carcinoma cell line (HeLa cells) from normal human peripheral blood cells has been achieved by exploiting their differential dielectric properties. The isolation process is carried out on a silicon chip containing a five-by-five array of microlocations. These microlocations contain underlying circular platinum electrodes with 80-micron diameters and center-to-center spacing of 200 microns. The surfaces of the electrodes and nonmetallized areas have been coated with a permeation layer to prevent the direct contact of cells with the electrode and also to minimize the nonspecific adhesion of the cells to the chip surface. An inhomogenous ac field is applied to the electrodes to create the conditions for dielectrophoretic separation of cells. Cell separation using dielectrophoresis as well as electronic lysis on a silicon chip would provide essential sample-processing steps which may be combined with a later multiplex electronic hybridization step in an integrated assay system.

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