Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-05
2003-05-13
Siew, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S287200, C436S536000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06562568
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns a process for preparing biological samples for the subsequent detection of an analyte, in particular a nucleic acid, in this sample. In addition reagent kits and new devices for sample preparation and new magnetic pigments are provided.
BACKGROUND ART
The sample preparation often has to meet special requirements in a method for the detection of an analyte in a biological sample. On the one hand, the analyte is often present at a very low concentration and, on the other hand, there are often many other substances in the sample which can interfere with the isolation or determination of the analyte.
WO 96/41811 discloses a process for the isolation of an analyte, especially a nucleic acid, from a biological sample wherein the sample which contains the analyte in a liquid is contacted with magnetic particles that have an outer glass surface which is essentially free of pores or has pores with a diameter of <10 nm, under conditions such that the analyte binds to the particle surface and the bound analyte is separated from the sample liquid. The process described in WO 96/46811 is very well suited to the purification of an analyte from a biological sample. However, it cannot be easily applied to an automated sample preparation.
Boom et al. (J. Clin. Microbiol. 28 (1990), 495-503) also describe a protocol for the purification of nucleic acids from a biological sample using silicon oxide particles fractionated according to size. However, this process is complicated and not suitable for automation and moreover there is a risk of carry-over.
In a method described in EP-A-0 757 106 for the extraction of nucleic acids, a sample is lysed, the nucleic acids present in the sample are bound to superparamagnetic metal particles, these are removed from the sample vessel with a pipette and thus separated from the other sample components. A disadvantage of this method is that losses may occur due to the necessity of having to remove the analyte from the sample with a pipette. Furthermore there is a risk of carry-over and contamination due to the use of several reaction vessels.
SUMMARY OF THE INVENTION
Hence the object of the present invention was to provide a new sample preparation process in which the disadvantages of the state of the art are at least partially eliminated. In particular it should be possible to automate the new process and have a temperature profile that is as simple as possible.
This object is achieved by a process for the isolation of an analyte from a biological sample comprising the steps:
(a) lysing the sample in a reaction vessel,
(b) adding a solid adsorption matrix
(c) incubating under such conditions that the analyte binds to the adsorption matrix,
(d) removing non-bound sample components from the reaction vessel,
(e) incubating under such conditions that the analyte is eluted from the adsorption matrix and
(f) separating the eluate from the adsorption matrix.
A further aspect of the present invention is a process for the isolation of an analyte from a biological sample comprising the steps:
(a) lysing the sample in a reaction vessel,
(b) adding a solid adsorption matrix,
(c) incubating under such conditions that the analyte binds to the adsorption matrix,
(d) separating non-bound sample components from the adsorption matrix,
(e) incubating under such conditions that the analyte is eluted from the adsorption matrix and
(f) separating the eluate from the adsorption matrix wherein at least steps (c) and (d) are carried out at essentially the same temperature.
DETAILED DESCRIPTION OF THE INVENTION
The process according to the invention is based on the selective binding of analytes to a solid adsorption matrix in the presence of a sample lysing buffer in which the analyte that is preferably a nucleic acid such as DNA e.g. chromosomal DNA, fragmented chromosomal DNA, plasmid DNA, viral DNA etc. or RNA e.g. mRNA, tRNA, rRNA or viral RNA etc., is separated from impurities of the sample such as proteins or cell debris. The sample can be any biological sample e.g. a body fluid such as blood, plasma, urine etc., a tissue sample, a sample of cultured cells or such like.
The adsorption matrix used in the process according to the invention is able to ensure the substantially selective binding of the analyte under the reaction conditions. A particulate adsorption matrix is preferably used which preferably contains a glass surface. Magnetic glass particles are particularly preferred, especially the magnetic particles described in WO 96/41811 with an external glass surface which is essentially free of pores or has pores with a diameter of less than 10 nm. Ferromagnetic particles are particularly preferred which have a particle size between 10 and 60 &mgr;m. Such particles can for example contain a core made of mica and magnetic particles immobilized thereon which is enclosed by a layer of glass. Whereas in WO 96/41811 the magnetic particles are placed in the individual reaction vessels in a solid form e.g. as tablets or a powder, the magnetic particles are preferably used according to the invention in the form of a suspension. Alcoholic suspensions having a concentration of about 5 to 20 mg/ml have proven to be particularly suitable. It was surprisingly found that, despite the high specific density of the magnetic glass particles, the suspension can be very reproducibly drawn out of a storage container which enables the process to be automated.
Although the glass particles described in WO 96/41811 give good results in the process according to the invention, particularly good results are obtained with glass particles whose glass phase contains the following metal oxides: SiO
2
, B
2
O
3
, alkali metal oxide e.g. K
2
O or/and Na
2
O and optionally Al
2
O
3
and an alkaline earth metal oxide e.g. CaO. The contents of these metal oxides are preferably as follows: 50 to 95 mol-% SiO
2
, 0.2 to 30 mol-% B
2
O
3
, 0 to 10 mol-% Al
2
O
3
, 0 to 20 mol-% alkaline earth metal oxide and 0.2 to 20 mol-% alkali metal oxide where the percentages are each based on the total weight of the glass phase.
A glass phase which contains SiO
2
, B
2
O
3
, K
2
O, Al
2
O
3
and CaO has proven to be particularly suitable for the isolation of RNA. A glass phase which contains SiO
2
, B
2
O
3
and Na
2
O has proven to be particularly suitable for the isolation of DNA.
In the process according to the invention the adsorption matrix is preferably added in an amount which corresponds to the minimum amount required to quantitatively bind the analyte present in the sample, in particular a nucleic acid, or the amount is somewhat larger, preferably at most 50% and particularly preferably at most 20% above this amount. The expected amount of nucleic acid in various type of samples can—if it is not already known—be determined in advance by common techniques e.g. phenol/chloroform extraction and subsequent measurement of the optical density.
Step (a) of the process according to the invention comprises lysing the sample in a reaction vessel. This lysis is usually carried out by lysing the cells present in the sample under denaturing conditions e.g. by adding a protease and a denaturing buffer. Proteinase K, pronase, elastase or/and lysozyme are preferably used as the proteinase. The use of proteinase K is particularly preferred.
The protease digestion is carried out in a denaturing buffer which contains a chaotropic compound e.g. urea or urea derivatives, preferably a chaotropic salt, particularly preferably a guanidinium salt such as guanidinium hydrochloride (especially for the isolation of DNA) or guanidinium thiocyanate (especially for the isolation of RNA) or a perchlorate or iodide. Concentrations in the range of 1 to 3 mol/l are preferred for guanidinium salts.
In contrast to the method described in WO 96/41811 for sample preparation, the solid adsorption matrix is only added after lysing the sample. This procedure results in a significantly lower unspecific binding of undesired sample components, e.g. proteins, to the adsorption matrix.
According to step (c) the anal
Harttig Herbert
Kleiber Jörg
Markert-Hahn Christine
Doyle Charles M.
Jen George C.
Pennie & Edmonds LLP
Roche Diagnostics GmbH
Siew Jeffrey
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