Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-02
2002-03-12
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025410
Reexamination Certificate
active
06355792
ABSTRACT:
The invention relates to a process for the isolation and purification of nucleic acids from liquids with the aid of a solid carrier material.
An extensive prior art exists which describes the isolation of nucleic acids from body fluids. Older methods comprise several working steps: enrichment of the nucleic acid-containing cells or compartments, lysis thereof, separation of the protein and membrane fractions, precipitation of the purified nucleic acids for the removal of contaminating chemicals. More recent methods use solid-phase extractions, the nucleic acid-containing compartments being lysed under specific reaction conditions and DNA and RNA or their subpopulations being bound to the solid phase. Specific reaction conditions are high molar concentrations of specific chaotropic substances or of water-soluble organic solvents. High molecular weight, genomic DNA of eukaryotic cells can likewise be captured and purified by simple lysis of the cells by means of detergence and physical interaction of the long DNA threads with microparticles or large-pore filters. It is also known that purified nucleic acids bind to chromatographic material such as hydroxyapatite in aqueous medium and can be removed using relatively high molar phosphate buffer.
U.S. Pat. No. 5,234,809 discloses a process for the isolation of nucleic acids, the binding to a solid phase taking place in the presence of high concentrations of chaotropic substances such as guanidinium salts, sodium iodide, sodium thiocyanate or urea. The bound nucleic acids are washed with a wash buffer comprising a chaotropic substance. For the removal of the highly concentrated salts, washing is then carried out with an alcohol- and/or acetone-containing wash solution. After careful removal of the organic solvents, the nucleic acids are eluted with water or with buffer of low ionic strength. Similar processes are disclosed in EP 0 572 907.
According to WO 96/18731, the binding to the solid phase should take place under neutral buffer conditions in the presence of detergents. However, the process has the disadvantage that only long-chain DNA molecules such as genomic DNA of eukaryotic cells can be bound to the solid phase. Short DNA molecules and RNA molecules cannot be directly isolated under the buffer conditions described.
The described methods for the isolation and purification of nucleic acids have a number of disadvantages. Chaotropic substances in high concentrations, organic solvents and detergents have an inhibitory effect on subsequent molecular-biological reactions for whose purpose the nucleic acids are isolated. Intensive washing steps or drying steps are necessary in order to remove these inhibitors quantitatively. The described chemicals and washing steps are also a hindrance to automation plans with the aim of rapidly and reproducibly isolating gene material from a high number of samples.
WO 97/34909 discloses a process for the isolation of nucleic acids in which the nucleic acids from the sample are bound to an organic crosslinked polymer which has basic groups. For this binding step, additions of detergents, chaotropic substances or organic solvents are not necessary. However, binding takes place slowly and requires incubation times of one hour or more.
It is thus the object to make available a process for the isolation and purification of nucleic acids which can be carried out without interfering addition of the substances mentioned (e.g. chaotropic substances), and which requires a short incubation time. The isolated or purified nucleic acids (DNA and RNA) should be able to be employed for molecular-biological reactions immediately after elution from the solid phase.
It has been found that, when using inorganic oxide materials containing hydroxyl groups, binding of nucleic acids to these carrier materials is possible even without addition of chaotropic substances if the pH is lowered to 1 to 6 by the binding buffer. This binding takes place rapidly, i.e. within less than 15 minutes. After an optional washing step, the nucleic acid can be dissolved by means of an elution buffer having a pH of between 7.5 and 11 and is ready for further molecular-biological reactions.
The invention relates to a process for the isolation and purification of nucleic acids from liquid samples, characterized by the following process steps:
a) provision of a liquid sample which contains nucleic acids;
b) provision of a carrier material consisting of an inorganic oxide material containing hydroxyl groups;
c) dilution of the sample from step a) with a binding buffer;
d) treatment of the sample from step c) acidified by means of a binding buffer with the carrier material from step b), the nucleic acids being bound;
e) separation of the sample and of the binding buffer after binding of the nucleic acids;
f) elution of the nucleic acids bound in step d) by means of an alkaline solution.
In preferred embodiments, a washing step e1) is inserted after step e), the pH of the wash buffer used here being ≦6.5. In further preferred embodiments, the pH of the binding buffer is between 3 and 6 and of the elution buffer from 7.5 to 9. Preferred carrier materials are silica gel and hydroxyapatite.
The invention further relates to reagent combinations for the process according to the invention for the isolation and purification of nucleic acids from liquid samples. In preferred embodiments, these reagent combinations contain all constituents necessary for carrying out the process: carrier material, binding buffer, one or more wash buffers and elution buffers. However, it is also possible to supply individual components of these separately, or to leave the obtainment of these components to the user, such that reagent combinations, for example, without the wash buffer are also a subject of the invention. Thus a reagent combination according to the invention can also comprise two or three constituents selected from carrier material, binding buffer, wash buffer and elution buffer, in particular comprise, for example, carrier material and binding buffer. Reagent combinations for the process according to the invention can further additionally contain auxiliaries such as centrifuge tubes or metering aids.
In
FIG. 1
, the binding kinetics for the process according to the invention (curves (A) and (B)) are compared with a process corresponding to the prior art according to WO 97/34909 (curves (C) and (D)). For the measured results shown in curves (B) and (D), bovine serum albumin was added to the sample in order to simulate the use of the processes for protein-containing samples. Further experimental details are found in the description for Comparative Example A.
As carrier materials, suitable inorganic oxide materials containing hydroxyl groups are known from the prior art, for example from U.S. Pat. No. 5,234,809; these include, in particular, crystalline or amorphous modifications of SiO
2
and silicates, i.e., for example, silica gel, kieselguhr, silicate glasses or zeolites, furthermore also hydroxyapatites. Preferred carrier materials are crystalline or amorphous modifications of SiO
2
and silicates.
The carrier materials can be present in the form of beads, particles, sheets, gels, filters, membranes, fibres, in capillaries, strips, tubes, microtitre plates etc. Appropriate magnetic particles can also be used. The carrier materials can also be applied, for example, as a coating to vessels. An equilibration buffer used for the carrier material is preferably one of the binding buffers mentioned below.
The binding of the nucleic acids from the sample is carried out by means of simple lowering of the pH to below pH 6. For this, a binding buffer is used which can maintain a pH range from 1 to 6, preferably from 3 to 5. As is known, the purine bases of the nucleic acids have improved stability in the preferred pH range. Suitable buffers are, for example, formate, acetate, citrate buffers or other buffer systems which have adequate buffer capacity in the pH range mentioned.
The buffer concentration should be in the range from 10 to 200 mM, depending on the
Hendriks Robertus
Holschuh Karl
Michelsen Uwe
Katcheves Konstantina
Merck Patent Gesellschaft
Millen White Zelano & Branigan P.C.
Yucel Remy
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