Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-07-23
2004-04-13
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06720417
ABSTRACT:
The invention concerns a method for the isolation of nucleic acids from a sample and a suitable device for this.
The introduction of the polymerase chain reaction (PCR) and subsequent alternative amplification systems for nucleic acids enabled this genetic material to be used as a material for examination by diagnostic tests. This has resulted in new analytical opportunities above all for the diagnosis of hereditary diseases, predisposition for certain diseases and infectious diseases which, among others, allows an earlier detection of the condition.
In order to convert the genetic material into a suitable form for enzymatic amplification, it is always necessary to release it from the biological sample material. In addition the nucleic acid must be protected from degradation by nucleases in the biological material or the environment and from degradation by chemical reaction conditions (Fe
++
/DTT or &bgr;-mercaptoethanol; NaOH; heat). The greatest demands are made on the freedom from contamination of the biological sample and of the nucleic acid that is isolated from it. In particular it is necessary to prevent transfer from the environment, by laboratory staff and cross-contamination between samples. The nucleic acid should be present in a buffered, aqueous, substantially salt-free solution for the amplification.
Whereas PCR always uses very small amounts of analyte (pg
g range), special problems may require the processing of a larger amount of sample. In order to, for example, identify circulating tumour cells with a sensitivity of one tumour cell in a background of 10
7
-10
8
normal cells, the nucleic acid must for example be isolated from 10-20 ml of a blood sample. After homogenizing the sample, an aliquot of the isolated RNA can thus be examined for expression of a tumour-associated gene.
In addition to the classical methods of nucleic acid isolation by means of enzymatic, mechanical or chemical lysis of the sample material, subsequent extraction of the proteins and lipids by phenol and phenol/CHCl
3
and precipitation of the nucleic acid from the aqueous phase with ethanol or i-propanol (Sambrook, J., Fritsch E. F. and T. Maniatis Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989, 2nd edition, 9.16-9.23; Ausubel F. M. et al. Current Protocols in Molecular Biology, John Wiley & Sons, 1987, 2.1.1-2.4.5), several commercial kits have been developed in recent years especially for PCR sample preparation which utilize the property that nucleic acids bind to glass surfaces under chaotropic salt conditions which has been known since the end of the seventies (Vogelstein B. et al., Proc.Natl.Acad.Sci: USA, 76 pp 615-619 (1979)). Other constituents of biological material such as proteins, lipids or salts are not bound and are therefore separated. Centrifugation vessels with glass fleece inserts for sample volumes up to 200 &mgr;l or silica gel suspensions which allow a batch process are known. Furthermore multiple devices in a strip and 96-well microtitre plate format with glass fleeces inserted in the bottom are known which can be operated with the aid of a vacuum chamber that is mounted underneath as well as by centrifugation. In these methods there is a great risk of contamination since all the vessels are in contact via the airspace with vacuum suction as well as with centrifugation and contamination by aerosols can occur.
A modified method (Miller et al., Nucl. Acids Res. 16: 1215) uses a concentrated salt solution to precipitate proteins and other accompanying substances after lysing the sample material. The nucleic acids in the supernatant are then precipitated by ethanol and collected by centrifugation. After the nucleic acids have been dissolved, they can be used for the amplification.
The current approaches are either limited with regard to the size of the volume that can be processed or are more time consuming or require an additional step to precipitate the nucleic acids. A multiple application is time consuming and increases the risk of contamination and mistakes. Batch process can be fundamentally scaled up but even then they have a risk of contamination.
Hence the object of the present invention was to provide a simple method for isolating nucleic acids from larger sample volumes.
Hence the invention concerns a method for isolating nucleic acids from a sample by taking the sample up into a first vessel through an opening, closing the opening of the first vessel with a closing element which contains a nucleic-acid-binding, liquid-permeable material and which on the side facing the opening contains means for attaching the element on the first vessel and, on the other side of the material, contains means for attaching the element to a second vessel, and transferring the sample through the material into the vessel attached to the other side. The invention also concerns a device for carrying out this method.
The components of the device according to the invention and the individual phases of the method according to the invention are shown schematically and as an example in FIG.
1
.
A method for isolating nucleic acids is understood as a method in which nucleic acids in a sample are separated from other sample components. This is achieved by binding the nucleic acids to a nucleic-acid-binding material. After the binding, the liquid can be separated from the material containing the nucleic acids. In order to isolate particularly pure nucleic acids, it is possible to remove substances that may still be adhering by washing the material with a liquid. If desired the nucleic acids can be detached again from the nucleic-acid-binding material. The nucleic acids are bound or detached from the material under conditions that depend on the material used.
Nucleic-acid-binding materials are known to a person skilled in the art. The material can be particulate and also fibrous. If the material is composed of particles it has proven to be advantageous to immobilize these particles e.g. by placing them between small liquid-permeable plates e.g. fabrics or fleeces made of fibrous material such as cellulose or plastics which have such narrow pores that the particles are held between the plates. However, the nucleic-acid-binding material is preferably a fibrous material e.g. in the form of fabrics or fleeces. Suitable materials are for example known from methods for isolating nucleic acids with the aid of centrifugation tubes or multiple devices in a strip format. The nucleic-acid-binding material must have the property that the sample liquid can pass through the material without any additional action of force or by applying a force e.g. by applying pressure or underpressure. However, since in the present method the nucleic acids are not bound by filtration of the nucleic acids from the sample, but by a method which utilizes the affinity of nucleic acids for surfaces, it is possible to use a relatively coarse porous material. This facilitates the flow even of relatively viscous sample liquids.
The liquid-permeable material is able to bind nucleic acids but allows passage of other components dissolved therein such as proteins etc. In a first variant the nucleic acids can be bound sequence-specifically by capture probes attached to the surface of the material. The capture probes have a base sequence which can bind under hybridization conditions to a complementary base sequence in the nucleic acids to be isolated. The use of sequence-specific materials allows the selective isolation of nucleic acids having a particular sequence. A method for binding nucleic acids to peptidic nucleic acids on the surface of solids is described for example in WO 95/14708. In a preferred case the liquid-permeable material has a glass-containing surface. The property of being able to bind nucleic acids has been already known for a long time for particulate and fibrous materials. Thus the use of glass fleeces for isolating nucleic acids is described for example in DE-A-19512369.
Nucleic acids within the sense of the invention are understood as nucleic acids of any origin e.g. nucleic acids
Amick Marilyn
Crane L. E.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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