Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1986-12-16
1993-01-05
Niebling, John
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
204299R, G01N 2726, B01D 5702
Patent
active
051768054
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to methods of separating mixtures of charged macromolecules (which term includes charged molecular complexes) by electrophoresis.
In gel electrophoresis, for example in polyacrylamide gels, a primary objective is high resolution and for this reason the migrating zones need to be kept as sharp as possible. Zone spreading is due in part to diffusion, but we have found that particularly for large molecules in concentrated gels, another broadening effect can occur. This is because the migrating molecules enter pores in the gel from which they cannot escape except by the way in which they entered. The effect is that of a `lobster pot` entrapment. With the much larger nucleic acid molecules similar interactions lead to loss of discrimination between different sizes of molecules. The effect of a smooth direct current is to drive the molecules deeper into the trap.
The practical effect is that a sample placed at the origin is not carried forward completely, but a "trail" of entrapped material is left behind as the sample migrates through the gel, leading to diminution of the migrating zone of material. If the initial sample is small, the entrapped material can eventually comprise such a large percentage of the total material initially placed at the origin, that the quantity of material in the migrating zone becomes too small to be observable or analysable. With much more sensitive detection methods now being available, it is now practical and desirable to apply much smaller samples to the gel and hence the foregoing problem has assumed much greater significance.
Recently, it has become important to be able to separate very large molecules such as chromosomal-size DNA. Some measure of success has been achieved by the use of complex arrangements of multiple electrodes to apply to the gel electrical fields in intermittently variable transverse (orthogonal) directions, which in practice is difficult to put into effect especially where multiple samples are being analysed simultaneously on the same gel. Such a system has been described by Cantor et al in U.S. Pat. No. 4,473,452, and in Cell, 37 (1984) pages 67 to 75. Their system involves the use of multiple arrays of diode-controlled electrodes, to achieve the desired complex field effects without encountering problems due to short-circuiting between the different arrays of electrodes.
The present invention provides an electrophoretic method of separating charged macromolecules, in which method the macromolecules are induced to move through a gel or similar physically-impeding medium, in a predetermined direction away from an origin, under the influence of an electrical potential the polarity of which is intermittently reversed.
In one embodiment of the invention, the current reversal is parallel to the predetermined direction in which the macromolecules are induced to move.
By this surprisingly simply technique, which in its simplest embodiment can be achieved by the use of a single pair of electrodes in standard electrophoresis equipment, and with a conventional power source supplemented by an appropriate switching device, it is possible to achieve molecular separations at least as effectively as by the more complex electrode arrangements hitherto proposed (e.g. by Cantor et al), and indeed in some instances superior separations may be achieved. Hence in its simplest form, the benefits of the invention can be obtained using conventional electrophoresis equipment supplemented by a switching means capable of providing the necessary unbalanced current reversal.
In the separation of DNA, the importance and practical benefits of current reversal have very recently been independently verified by Olson et al, and was the subject of a lecture delivered by Olson in Heidelberg in November 1985. Furthermore, a disclosure by Cantor et al in Nature, 319 (1986) pages 701 to 702, suggests that in their multiple field approach, field shapes leading to angles between alternate fields that range from 100.degree. to 150.degree., enhance resolution and inhe
REFERENCES:
patent: 3506554 (1970-04-01), Broome
Hoefer Scientific Instruments
Niebling John
Starsiak Jr. John S.
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