Separation of Large DNA Molecules by Contour-Clamped Homogeneous Electric Fields

Abstract

Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.

High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1‐independent DSB repair pathway

“…A Biorad CHEF gel apparatus was used to separate DNA in plugs using previously described conditions (Chu et al 1986). …”

A physical map around the WAGR complex on the short arm of chromosome 11

“…One percent agarose gels were run· in an LKB pulsaphor unit in 0.5X TBE using the double inhomogeneaus field electrodes (Schwartz and Cantor, 1984;Carle and Olsen, 1984) or the CHEF electrode array described by Chu et aL (1986). In some experiments the Rotaphor electrophoresis unit (lmmunetics, Cambridge, MA) was used.…”

Chromosomal Circularization inStreptomyces griseusby Nonhomologous Recombination of Deletion Ends

“…S. griseus strains were reciprocally cultured at 289 C in a 500-ml Sakaguchi ‰ask containing 100 ml of GMP medium and total DNAs were isolated as described by Suwa et al 17) Gel samples for PFGE were prepared by the mycelium method as described previously, 2,18) digested in gels, and separated by contour-clamped homogeneous electricˆelds (CHEF). 19) CHEF was done in 0.5×TBE buŠer using 1.0z agarose at 159 C. E. coli strains were cultured at 379 C in LB medium, and cosmid and plasmid DNAs were extracted as described by Sambrook et al 20) Southern hybridization. DNA fragments were separated by CHEF or conventional agarose gel electrophoresis and transferred to nylon membranê lters by the capillary method.…”