Chromatography material

Details Chromatography material Chromatography material

1996-10-23

1998-12-01

Therkorn, Ernest G.

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

210656, 210657, 210658, 2101982, 536 254, B01D 1508

Patent

active

058433120

DESCRIPTION:

BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 of PCT/EP95/00434 Feb. 7, 1995.
The invention concerns a chromatography material for separation of nucleic acid mixtures, as well as a chromatographic method for separation of nucleic acid mixtures.
Progress in biochemistry, molecular biology and human genetics and its applications in technology, medicine, pharmacy and genetic engineering require rapid and systematic separation and isolation of nucleic acids. For example, a common problem in molecular biology is that a specific nucleic acid must be isolated from a naturally occurring mixture of a hundred or more components, which is contained in this mixture in concentrations of less than 0.1%. The requirements on a chromatographic process therefore include, on the one hand, quantitative isolation of the nucleic acid and, on the other hand, quantitative separation of 99.9% impurities in order to purify the nucleic acid as molecular species up to homogeneity.
The known chromatography methods, however, are not satisfactory with respect to the attainable resolution of the nucleic acid mixture and the related purity of the nucleic acid being isolated. In known methods costly and cost-intensive equipment, like high-performance liquid chromatographs (HPLC) and/or ultracentrifuges, toxic substances, like phenol, chloroform or ethidium bromide, or substances that interfere in subsequent experiments, like RNAse or protease, are also frequently used.
A chromatographic support material from silica gel suitable for separation of proteins, peptides and nucleic acids is described in U.S. Pat. No. 4,029,583, to which a stationary phase with groups that form anion or cation exchangers is bonded by means of a silanization reagent, these groups interacting with the substances being separated. The silanized silica gel is brought into contact with water, during which there is a hazard that the stationary phase will polymerize and the chromatography material will become unusable.
According to EP-B 0 104 210 nucleic acid mixtures can be separated into their components if a chromatography material whose support is first converted with a silanization reagent having a flexible chain group is used, which in turn is again converted to a finished chromatography material by reaction with a reagent that forms an anion or cation exchanger. This known chromatography material separates nucleic acid mixtures using HPLC equipment. However, in long-chain nucleic acids the use of HPLC can lead to damage (for example degradation) of long-chain nucleic acids due to high shear forces.
A process for separation of long-chain nucleic acids is described in EP-B 0 268 946 in which long-chain nucleic acids are fixed on a porous anion exchanger. The anion exchanger described in EP-B 0 104 210 is used as anion exchanger. Satisfactory purity of the isolated nucleic acids, however, cannot be achieved.
A silanized chromatographic support is described in DE-A 39 35 098 in which the silanization reagent has at least one reactive group already converted with a primary or secondary hydroxylamine or contains a reactive group that can be converted with a hydroxylamine. Separation of nucleic acids in HPLC using a continuous salt gradient has been demonstrated with this material (example 5).
However, the separation performance of known materials is not sufficient to solve difficult separation problems with simple chromatographic methods that operate with a specified number of theoretical plates, like simple column chromatography in which flow of the mobile phase is produced by shear force, separations in spin columns (to be used in a centrifuge), in which flow of the mobile phase is produced by centrifugal force, or batch methods in which the chromatography material is in suspension and with avoidance of a continuous salt gradient, i.e., using a step gradient. In particular, simple column chromatographic methods according to the prior art often produced incomplete separations during isolation of DNA from cultures of transformed bacteria in which ver

REFERENCES:
patent: 4029583 (1977-06-01), Ho Chang
patent: 4049496 (1977-09-01), Henry
patent: 4108603 (1978-08-01), Regnier
patent: 4298500 (1981-11-01), Abbott
patent: 4699717 (1987-10-01), Riesner
Snyder, Introduction to Modern Liquid Chromatography, John Wiley & Sons, Inc, 1979, New York, pp. 484-485 & 493-494.
Snyder, Introduction to Modern Liquid Chromatography, John Wiley & Sons, New York, 1979, p. 838.

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