Nucleic acid isolation

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C536S023100

Reexamination Certificate

active

06737235

ABSTRACT:

The present invention belongs to the field of purification of nucleic acids, in aqueous medium.
A process is known according to the document WO-A-95/04140 for purifying, in aqueous medium, nucleic acids present in a sample, according to which said sample is brought into contact with a particulate system consisting of silica beads, in the presence of a chaotropic substance, and then the nucleic acids attached to the beads are separated from the final aqueous solution.
In accordance with the document F. Meunier et al., Polymers for Advanced Technologies, Volume 6, pp. 489-496, (1995), the preparation of a polymer called PNIPAM, by polymerization of (1) N-isopropylacrylamide, (2) N,N-methylenebisacrylamide and (3) 2-aminoethylmethacrylate chloride, in the presence of a polymerization initiator, is described. The behavior of this surface-functionalized polymer can make it particularly suited to a covalent attachment of biological molecules.
The document EP-A-0 161 881 teaches that a heat-sensitive polymer such as the polymers obtained by copolymerization of monomers of N-alkyl- or of N-alkylene-acrylamide or methacrylamide and of monomers of acrylic or methacrylic derivatives, can be used in the isolation of biological material, by virtue of its capacity to change structure as a function of the temperature. It has an open structure at low temperature, which facilitates the attachment of a biological material, and a retracted structure at high temperature, which allows the liberation of the attached biological material. The control of the steps of attachment and liberation of the biological material can therefore be performed by varying the temperature. For a better control, it is possible, in addition, to vary the pH.
The use proposed by this document extends to the isolation of any biological material present in a sample, and in particular nucleic material and protein material, without any specificity.
According to the invention, a process for the selective isolation of a nucleic material present in a sample is provided. Even if the sample is complex and contains a protein material and/or inhibitors of enzymatic reaction, the process of the invention limits or even eliminates any isolation of the protein material and/or of said inhibitors, while promoting the isolation of the nucleic material.
A process for the isolation in aqueous phase, according to the invention, of a nucleic material present in a sample, comprises the following steps:
according to a so-called step (a) for producing the adsorption reagent, an adsorption reagent is available which comprises a sol consisting of an aqueous continuous phase and a discontinuous phase of the particulate support which comprises a functionalized, particulate polymer, said polymer being obtained by polymerization of (1) a first water-soluble monomer of acrylamide or of an acrylamide derivative, (2) at least one cross-linking agent and (3) at least a second cationic and water-soluble functional monomer, said polymer having a predetermined lower critical solubility temperature (LCST) which is between 25 and 45° C., preferably between 30 and 40° C., according to a so-called step (b) for bringing into contact, the adsorption reagent is brought into contact with the sample containing the nucleic material,
according to a so-called adsorption step (c), for the bringing into contact according to (b), at least one and preferably at least two of the following parameters for the reaction medium are chosen:
pH at most equal to 7,
ionic strength at most equal to 10
−2
M,
temperature less than the LCST of the polymer,
according to a so-called separation step (d), after having optionally observed that the adsorption has taken place, the discontinuous phase and in particular that having adsorbed the nucleic material are separated from the continuous phase,
according to a so-called desorption step (e), the nucleic material is dissociated, by desorption, from the particulate support by increasing the ionic strength up to an ionic strength greater than 10
−2
M.
Advantageously, for the desorption step (e), at least one of the parameters selected from the pH and the temperature is in addition varied as follows:
increase in the pH up to a pH greater than 7,
increase in the temperature up to a temperature greater than the LCST of the polymer.
The invention also relates to a process for the isolation, in aqueous phase, of a nucleic material present in a sample, comprising a step of adsorption of said nucleic material, onto a particulate support, allowing a use as such of the nucleic material adsorbed onto the particulate support, in a subsequent analytical step. This process comprises the following steps:
according to a so-called step (a) for producing the adsorption reagent, an adsorption reagent is available which comprises a sol consisting of an aqueous continuous phase and a discontinuous phase of the particulate support which comprises a functionalized, particulate polymer, said polymer being obtained by polymerization of (1) a first water-soluble monomer of acrylamide or of an acrylamide derivative, (2) at least one cross-linking agent and (3) at least a second cationic and water-soluble functional monomer, and said polymer having a predetermined lower critical solubility temperature (LCST) which is between 25 and 45° C.,
according to a so-called step (b) for bringing into contact, the adsorption reagent is brought into contact with the sample containing the nucleic material,
according to a so-called adsorption step (c), for the bringing into contact according to (b), an ionic strength at most equal to 10
−2
M is selected for the reaction medium,
according to a so-called separation step (d), after having optionally observed that the adsorption has taken place, the discontinuous phase is separated from the continuous phase, according to which process the desorption step is optional.
In accordance with a preferred embodiment of the latter process, according to the adsorption step (c), for the bringing into contact according to (b), at least one of the following parameters is in addition selected for the reaction medium:
pH at most equal to 7,
temperature less than the LCST of the polymer.
Of course, this process may comprise, after the separation step (d), a so-called desorption step according to which the nucleic material is dissociated, by desorption, from the particulate support by varying at least one of the parameters selected from the ionic strength, the pH and the temperature, as follows,
increase in the ionic strength up to an ionic strength greater than 10-
2
M
increase in the pH up to a pH greater than 7,
increase in the temperature up to a temperature greater than the LCST of the polymer.
At least the ionic strength is advantageously varied.
The processes defined above according to the invention will be preferably carried out according to two variants related to step (a).
According to a first variant which will be illustrated in the examples, the particulate support consists of said particulate polymer, and in this case, the cross-linking agent(s) (2) are water-soluble.
According to a second variant, the particulate support comprises, in addition, an organic or inorganic core, completely or partially coated with said particulate polymer, said core not modifying the adsorption properties of the polymer in relation to said nucleic material. The core or core portion then fulfills the function of the cross-linking agent (2), it being possible to provide another cross-linking agent of the water-soluble cross-linking agent type. By way of example, the core may be a polystyrene core, and/or comprise a magnetic compound.
According to a specific and preferred embodiment of these processes, at least one probe and/or one primer capable of specifically hybridizing to the nucleic material before or after step (b) is added to the sample before step (b), or to the reaction medium after step (b), and in particular after step (c) or step (d).
In another specific embodiment, the nucleic material consists of a probe or a primer, and accordi

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