Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-08-07
2004-10-05
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S306100
Reexamination Certificate
active
06800752
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a chromatography material for separation of nucleic acid mixtures and a method of separating nucleic acid mixtures using this chromatography material.
BACKGROUND
The use of chromatography and the chromatography materials used in this process have become indispensable in such fields as biochemistry, medicine, pharmacy and genetic engineering. With the help of chromatography materials, biomolecules such as nucleic acids and proteins are rapidly and systematically separated and isolated. In molecular biology, it is often necessary to isolate certain nucleic acids, which are present in this mixture in concentrations of less than 0.1%, from a naturally occurring mixture of more than a hundred different components. The requirements of a chromatographic method and the chromatography material used in the method therefore include, first, quantitative isolation of the nucleic acids, and secondly, quantitative separation of impurities to thereby purify the nucleic acid as a molecular species until it is homogeneous for subsequent analysis.
In the known chromatography methods, inorganic granular chromatography materials having defined particle and pore sizes are used, their surface having been modified with a silanizing reagent to produce a stationary phase. The reaction with a reagent forming an anion or cation exchanger then leads to the finished chromatography material.
For example, International Patent WO 91/05606 describes a carrier material for chromatography which is suitable for separation of various species of nucleic acids. Suitable carriers include silica gel, aluminum oxide, titanium dioxide, porous glass or resins. The carrier material should have a particle size of 3 to 500 &mgr;m with a pore size of approximately 10 to 1000 nm and a specific surface area of 5 to 800 m
2
/g. The surface of this granular support is silanized with alkoxysilanes. Thus, in addition, a chromatographic carrier material is described, which is based on silica gel and having anion exchanger groups that are obtained by reacting the alkoxysilane with a secondary hydroxylamine.
Thus, according to European Patent 0 104 210, silica gel materials have a particle size of 3 to 100 &mgr;m, a void size of 10 to 1000 nm and a specific surface area of 5 to 800 m
2
/g are described. These materials are then treated at the surface with a silanizing agent and used with ion exchanger functions in chromatography for separation of nucleic acids.
Finally, European Patent 0744025 discloses a chromatography material in which a carrier of silica gel is reacted with a silanizing reagent, whereby carrier materials having a pore diameter of 4 to 6 nm are selected. The particle size of the carrier is 1 to 500 &mgr;m.
In chromatographic separation of nucleic acid mixtures using conventional granular chromatography materials, it has been found that it is very time-consuming to perform such separations. For example, if a certain column was used with a modified silica gel as the carrier for chromatography, it would be expected that an adsorption time of the nucleic acids on the carrier of at least 20 minutes under gravity flow conditions would have to be accepted.
The chromatography materials known in the past have also had a defined porosity, because according to the prevailing opinion only a porous support, i.e., one with an enlarged surface area, would guarantee sufficient loading with ion exchanger. However, this porosity has the disadvantage that the quality of nucleic acid separation is not optimum. This is attributed to the fact that other substances present in the mixture during separation, e.g., RNA and proteins, diffuse into the pores and thus have a negative effect on the separation process.
Thus, would be advantageous to make available a chromatography material that could perform a separation of nucleic acid mixtures within the shortest possible period of time, while at the same time achieving an excellent resolution of the nucleic acid mixtures and thus an excellent purity of the nucleic acids isolated.
It would also be advantageous to make available a method with which a nucleic acid mixture can be separated with the highest possible resolution and purity of the individual components in the shortest possible amount of time.
SUMMARY OF THE INVENTION
The present invention relates to a chromatography material for separating nucleic acid mixtures, having a carrier and ion exchanger functions applied to it, wherein the carrier is a fibrous material.
This invention relates to a method of separating nucleic acid mixtures with the chromatography material according to this invention, where the chromatographic separation of the nucleic acids is performed by the action of a force.
The subclaims concern preferred embodiments of the method according to this invention.
This invention also concerns a kit for separating nucleic acid mixtures which contains a chromatography material according to the present invention. The kit includes the chromatography material according to this invention in the desired arrangement together with corresponding buffers for performing the chromatographic separation of nucleic acid mixtures using the chromatographic material.
REFERENCES:
patent: 4780369 (1988-10-01), Schnabel et al.
patent: 5451370 (1995-09-01), Jones
patent: 36 09 021 (1986-09-01), None
patent: 43 34 359 (1995-04-01), None
patent: 000443734 (1991-08-01), None
patent: WO 83/03363 (1983-10-01), None
patent: WO 95/22053 (1995-08-01), None
Bart et al. “Continuous Separation of Carbohydrates by the Use of Annular Chromatography”, 1998, Wissenschaftliche Kurznitteilungen, pp. 142-144.
Dr. Tittgen Biotechnologie
Ketter James
Piper Rudnick LLP
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