Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1991-02-27
1992-04-28
Niebling, John
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
2041831, 204299R, G01N 2726
Patent
active
051085674
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an electrophoresis method and apparatus which may be used in conjunction with conventional electrophoretic media, such as agarose gels, for the separation of large molecules, and in particular large DNA fragments.
In conventional electrophoretic techniques, DNA molecules above a certain length display uniform velocity and therefore are not resolved. As early as 1982 it was shown that large DNA fragments can be separated, according to size, in conventional agarose gels by applying periodically alternating electric field vectors (Schwartz et al., 1982; Schwartz and Cantor, 1984). Several different pulsed field systems now achieve resolution of DNA fragments in the range 50 kilobases to at least 12.6 megabases (Mb) (Orbach, 1988). Whilst the theoretical basis for large fragment separation by pulsed field systems remains largely speculative it is thought that the pulsed field systems obtain this separation by forcing the molecules to reorient cyclically. The available systems vary considerably in their box design, gel dimensions, electrode configuration, pulsing algorithm, run time requirements, separation power, inter-lane comparability, ease of handling, and versatility (Cantor et al., 1988).
The pulsed field systems of the prior art may be divided into two types. The first type comprises systems in which the electric field vectors of the pulsed field system are oriented in a single plane defined by the agarose gel. In such systems, the separation of molecules in each lane of the gel is achieved by the alternating electric fields forcing the molecules to reorient cyclically within the plane of the gel. The gel, supported within a gel tank, is generally oriented with the plane of the gel horizontal, and two sets of electrodes positioned at a predetermined angle.
Examples of such systems include orthogonal field agarose gel electrophoresis (OFAGE), horizontal and vertical field inversion gel electrophoresis (FIGE), rotating gel/electrode electrophoresis (RGE/REE), and contour-clamped homogeneous electric field gel electrophoresis (CHEF) and its variants, e.g. programmable, autonomously controlled electrode gel electrophoresis (PACE).
OFAGE is the archetypal system using either mixed uniform
on-uniform or non-uniform field configurations (Schwartz and Cantor, 1984; Van Ommen and Verkerk, 1986). It suffers, however, from marked distortion of the outer lanes of the gel, thereby limiting the area of the gel usable for the sizing of molecules and inter-sample comparison, to a few central lanes.
FIGE is a system based on reversing the polarity between only two parallel electrodes at either end of the gel and creating a net movement by using non-identical forward and reverse pulse times in each cycle (Carle et al., 1986). It is capable of producing straight lanes on large gels. However, the 180 degree angle between pulse vectors results in unacceptable broadening of bands on prolonged runs and thus compromises resolution, especially of large molecules. In the vertical variant of this system, the vertical orientation of the plane of the gel ensures more effective cooling of the gel than in the horizontal variant (Dawkins et al., 1987). The vertical system still suffers, however, from the inherent broadening of bands and low resolution of FIGE.
RGE/REE are systems in which either a circular gel or a single pair of electrodes are periodically rotated with respect to the other (Serwer, 1987; Southern et al., 1987). The molecules to be separated are thus exposed alternately to two uniform electric fields at a fixed angle. Although the systems give straight lanes over a fairly large area, their major drawback lies in their reliance on moving parts, which, given the long run times and extended day to day use, makes them liable to mechanical breakdown.
CHEF is a system based on a hexagonal array of up to 24 electrodes clamped to predetermined electric potentials (Chu et al., 1986; Vollrath and Davis, 1987). This design and its variants (Clark et al., 1988; Birren et al., 1988) involve the most compl
REFERENCES:
patent: 3423306 (1969-01-01), Hurwitz et al.
patent: 3520793 (1970-07-01), Kolin
patent: 3930982 (1976-01-01), Batha et al.
patent: 4693804 (1987-09-01), Serwer
patent: 4740283 (1988-04-01), Laas et al.
Medical Research Council
Niebling John
Ryser David G.
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