Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-26
2001-04-17
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S006120, C435S094000, C436S501000, C422S051000, C422S051000, C422S051000, C422S051000, C422S068100
Reexamination Certificate
active
06218116
ABSTRACT:
The present invention relates generally to the treatment by complexing of a liquid medium, comprising one or more components determining the presence, in said liquid medium, of a plurality or multiplicity of different ligands in the free state.
In the present description, and the claims, the following definitions are adopted below.
“Ligand” is understood to mean any entity or molecule, of a chemical, biochemical or biological nature, capable of binding in a specific manner to another entity or molecule, called “anti-ligand”, through one or more noncovalent bonds.
Emerging in particular from this definition are different biological or biochemical entities, in particular biopolymers, capable of forming a complex with each other and in a complementary manner:
they represent, for example, a polypeptide, in particular an antibody (ligand) capable of specifically binding to an epitope or antigen (anti-ligand), or conversely
they also represent, as shown by way of example in the description below, any nucleic acid or nucleic material capable, in single-stranded form, of binding in a complementary manner to an oligonucleotide or a polynucleotide.
The reaction or equilibrium of a chemical nature, by which the ligand binds, or pairs, or forms a complex with the anti-ligand, will be designated hereinafter by the term “complexing”. The product of this reaction or equilibrium will be designated hereinafter by the term “complex”. In the case of a nucleic material or macromolecule, the complex will be designated hereinafter by the term “duplex”. “Nucleic material” or “nucleic acid” is understood to mean any macromolecule comprising at least one unbranched, linear chain of nucleotides, which are themselves modified or otherwise. Emerging from this definition are, quite obviously, any deoxyribonucleic acid, modified or otherwise, and any ribonucleic acid, modified or otherwise. Such a macromolecule may be single-stranded, or double-stranded, or may have any other secondary or tertiary conformation. In practice, in the description hereinafter and by way of example, this nucleic material will be the one directly subjected to the treatment, or the one from which a liquid medium is obtained, containing the ligand(s), so as to then be treated in accordance with the present invention.
“Oligonucleotide” is understood to mean any polynucleotide, comprising at least five monomers, and preferably at least seven, for example eight monomers, which consist of a natural nucleic acid, or a nucleic acid modified on at least one of its constituent components, namely on the sugar, the nitrogenous base or the phosphate group.
“Support” is understood to mean any substrate at the surface of which anti-ligands may be permanently immobilized or attached, directly or indirectly, both covalently and noncovalently.
The substrate is either self-supporting, for example a sheet of an inert and transparent material, or is itself supported by a rigid base, for example a layer of acrylamide on a glass slide.
“Surface” is understood to mean the surface of the substrate accessible to the anti-ligands, whether it is a solid substrate or a porous substrate, in which case the true surface developed is greater than the apparent surface.
In the description hereinafter and by way of example, the oligonucleotides will constitute the anti-ligands defined above.
The complexing reaction between a nucleic material or a nucleic acid, on the one hand, and a complementary oligonucleotide, on the other, may be designated hereinafter by the term “hybridization”.
In accordance with the document WO-92/10558 and WO-93/22680, and according to a so-called “reverse dot-blot” bioassay format, a method and a device are described for treatment by complexing, namely hybridization, of a liquid medium, in the case of a nucleic sample, comprising one or more nucleic components, determining the presence in this liquid medium of a plurality of different nucleic fragments, segments or sequences (ligands) in the free state. The principal application of this treatment is the sequencing, by hybridization, of the nucleic sample according to known principles and methods disclosed in documents (6) and (7).
According to this method:
a) a support for plural complexing, of the biochip type, is provided on which there are distributed a plurality of discrete coupling sites or zones, separated from each other, in which there are immobilized a plurality of oligonucleotides (anti-ligands) which are respectively different and capable of being complexed with the nucleotide sequences complementary to the nucleic fragment(s), respectively;
b) under isothermal conditions from one coupling site to another, the liquid medium, or nucleic sample, is brought into contact with this complexing support, whereby the free nucleotide sequences pair with the fixed oligonucleotides respectively, and are as a result attached to the plural complexing support;
c) a parameter representative of the presence and/or of the quantity of the different complexes or hybrids obtained is observed at the different coupling sites of the plural complexing support, so as to generate signals and/or information which are representative of the presence and/or of the quantity of the different nucleotide sequences in the original liquid medium; preferably, these complexes are observed using labeling of the component(s) of the nucleic sample, or labeling of the different hybrids which are obtained and which are attached to the support.
As stated above, this process works under completely isothermal conditions, that is to say by providing the same hybridization temperature from one coupling site to another, at the time of bringing the liquid medium into contact with the plural complexing support, and/or at the time of washing this support after hybridization.
In accordance with the document EP-0,535,242, a similar method has been described and proposed which also works under isothermal conditions, and according to which at each coupling site, the concentration of corresponding oligonucleotide specific for said site is chosen as a function of the working temperature, so as to promote the stability of the corresponding complex obtained with the complementary nucleotide sequence.
According to this document, this concentration is chosen by observing the percentages of complementary nucleotide sequence eluted at each coupling site, during a washing phase which is performed with a washing liquid whose temperature is varied and which is incorrectly termed heat gradient washing.
The solutions described above are subject to a technical difficulty which is identified in the document WO-92/10588 but which has not been solved. Indeed, at a predetermined hybridization temperature, and for a given length of sequence of the oligonucleotide (anti-ligand), the stability of the complex obtained by hybridization of a nucleic acid probe with its target complementary sequence varies according to this sequence. Thus, two oligonucleotides differing from each other only by one of the nucleotides, namely T in one case and G in the other case, although of the same length and of a similar sequence, do not hybridize their respective complementary nucleic acids with the same stability. In other words, since the stability of the complexes obtained depends on the temperature, the sample nucleic acid should be hybridized with the oligonucleotide probes, carried on the plural complexing support, at a sufficiently cold temperature for the least stable of the possible complexes to form, otherwise false-negatives will be obtained after hybridization, that is to say oligonucleotide probes generating no hybridization signal although their complementary sequence is present in the sample. However, by carrying out the procedure in this manner, there are instances in which oligonucleotide probes form complexes which are sufficiently stable with sequences which are not perfectly complementary; in such a case, false-positives are then obtained, that is to say oligonucleotide probes generating a hybridization signal although their complementary sequences ar
Bio Merieux
Horlick Kenneth R.
Oliff & Berridg,e PLC
Taylor Janell E.
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