Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-01-11
2003-03-25
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C536S022100, C536S025300
Reexamination Certificate
active
06537783
ABSTRACT:
The present invention relates to a process for amplifying a sequence of a target nucleic acid.
The document EP-A-0 285 057 discloses a process for treating a nucleotide, consisting in introducing, into one of the constituent elements of said nucleotide, namely the sugar, the purine or pyrimidine base and the phosphate groups, a functional group having various uses, in particular those of labeling. The resulting nucleotide, which has thus been treated, can, according to this document, be incorporated into a polynucleotide, in particular in double-stranded form, without the double helix being destabilized by the presence of this nucleotide.
However, in this case, the functional groups which are attached to the nucleotide in accordance with this prior art exhibit a variety of phenomena, such as steric hindrance, hydrophobic interactions or complexing phenomena, which prevent the polynucleotide which has incorporated said treated nucleotide from being recognized by the majority of the enzymes which would recognize the corresponding polynucleotide into which said treated nucleotide had not been incorporated.
The document WO-A-92/00989 proposes a process which uses a protein for labeling a nucleic acid sequence, in particular for obtaining a labeled probe, by amplifying a target nucleic acid. According to this process, use is made of modified nucleotides which differ from the natural nucleotides by the presence of a reactive function, amplification is carried out in order to obtain a prefunctionalized probe, and the resulting probe is reacted, by way of the reactive functions, with a protein label. To this end, the reactive function of the modified nucleotide is a nucleophilic function which is selected from thiol functions, which may, where appropriate, be protected, and amino functions.
The drawback of this process lies in the selected label which, because of its size, with the molecular weight being of the order of several thousand, will substantially limit the rate and/or yield of the covalent coupling between said label and said function, with this problem increasing with the number of modified nucleotides which are incorporated into the probe. What is more, the label can lead to a deceleration of the reactions in which the resulting probe is involved, in particular in a hybridization.
The present invention provides an amplification process which overcomes the previously mentioned drawbacks and which, in particular, does not disrupt the incorporation of the nucleotides and, as a consequence, does not have a significant influence on the yield and/or the sensitivity in a target amplification reaction, and which, furthermore, makes it possible to obtain excellent labeling of the amplification products by avoiding label instability phenomena which were previously associated with incorporation of the label.
Thus, the present invention relates to a process for amplifying a target nucleic acid sequence, according to which:
at least the following are available: the sequence of a target nucleic acid, at least one oligonucleotide primer which is specific for the target sequence, and one or more nucleotide enzyme activities,
the target sequence is amplified under nucleotides is a prefunctonalized nucleotide which differs from the other nucleotides at least by the presence of at least one covalently reactive function, which is unprotected and which is arranged in at least one predetermined site on the base of said nucleotide, in order to obtain a prefunctionalized amplification product which includes at least one said prefunctionalized nucleotide.
According to an advantageous process of the invention, this process additionally comprises the following steps:
a reagent is available which comprises a covalently anti-reactive function, which is specific for the reactive function of the prefunctionalized nucleotide, and a functional group, and
the prefunctionalized amplification product is reacted, directly or indirectly, with the reagent in order to obtain a functionalized amplification product.
Some of the terms employed in the present description are defined below, after which the invention is explained in detail.
Nucleotide according to the invention is understood as being a natural or modified nucleotide monomer as defined below.
Thus, the nucleotide monomer can be a natural nucleic acid nucleotide whose constituent elements are a sugar, a phosphate group and a nitrogen base; the sugar is ribose in RNA and is 2′-deoxyribose in DNA; depending on whether the nucleic acid is DNA or RNA, the nitrogen base is selected from adenine, guanine, uracil, cytosine and thymine; or a nucleotide which is modified in at least one of the three constituent elements; by way of example, the modification can take place at the level of the bases, generating modified products such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other modified base which permits hybridization, at the level of the sugar, namely, for example, replacement of at least one deoxyribose by an analog (for example: P. E. Nielsen et al., Science, 254, 1497-1500 (1991)), at the level of the phosphate group, for example boronate, alkylphosphonate or phosphorothioate derivatives.
A protective group is understood as being the groups which are conventionally employed in the chemical synthesis of nucleosides, nucleotides and oligonucleotides (see, for example: Chemistry of Nucleosides and Nucleotides, edited by Leroy B. Townsend, Plenum Press, New York and London and Protocols for Oligonucleotides and Analogs, Synthesis and Properties, edited by Sudhir Agrawal, Humana Press, Totowa, N.J.).
A labeling functional group of the invention is a molecule which is capable of directly or indirectly generating a detectable signal. The group is, in particular, selected from radioactive isotopes, enzymes which are selected, in particular, from peroxidase, alkaline phosphatase and b-galactosidase, and those enzymes which are capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chromophoric, chromogenic, fluorophoric, fluorigenic or luminescent chemical compounds, nucleotide base analogs and ligands such as biotin.
When the label is unable to generate a signal directly, for example when the label is an enzyme, it is necessary to add a visualizing substance, for example a substrate which corresponds to the enzyme, with the enzyme/substrate reaction generating a detectable complex for example a chromogenic or luminescent compound. By way of example, the visualizing reagent can be ortho-phenylenediamine or 4-methylumbelliferyl phosphate.
The covalently reactive function of the prefunctionalized nucleotide and the anti-reactive function of the reagent are electrophilic and nucleophilic organic chemical functions, respectively, or vice versa.
The electrophilic organic chemical function is advantageously selected from the aldehyde, activated ester, carboxylic acid, isothiocyanate, haloacyl derivatives and sulfonyl chloride functions.
The nucleophilic organic chemical function is advantageously selected from the amino, thiol, oxyamino, hydrazine and hydrazide functions; it is preferably the alkoxyamino function.
According to one variant of the invention, the covalently reactive function of the modified nucleotide is attached to the base by way of a coupling arm and/or the covalently anti-reactive function of the reagent is attached to the functional group by way of a coupling arm.
The coupling arm is selected, in particular, from saturated or unsaturated hydrocarbon chains, which are interrupted, where appropriate, by amino, amido and oxy functions.
According to a preferred process, the covalently reactive function is the oxyamino function and the covalently anti-reactive function of the reagent is the aldehyde function, and this latter is linked to a labeling functional group such as a fluorescent or luminescent group.
Advantageously, the aldehyde function is linked to the functional group by the coupling arm —NH—CS—NH—(CH
2
)
3
—, and the functional gr
Defranc Eric
Guillou-Bonnici Françoise
Hoang Antoine
Laayoun Ali
Lhomme Jean
Bio Merieux
Marschel Ardin H.
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