Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-12-03
2001-04-03
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C536S023100, C536S024300, C536S024330, C436S094000
Reexamination Certificate
active
06210932
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to means for detecting nucleic acids by hybridization which can be used, in particular, for diagnosis.
BACKGROUND OF THE INVENTION
The techniques for identifying RNA or DNA sequences (target sequences) in a sample involve hybridizing the target sequence with a labelled probe and detecting the duplex which has been formed, after the unhybridized probe has been removed.
These techniques are commonly used, in particular, for diagnosing viral or infectious diseases, for medical or genetic research, for identifying clones, for analysing transcribed genes, etc.
Two types of probe are employed, i.e. so-called long probes (generally of a size larger than 100 bases) and short probes, whose sizes vary between 10 and 30 bases in length. The probes which are most commonly used in medical diagnosis are synthetic oligonucleotides which are coupled to a label, either by direct attachment to this label or by coupling to a ligand; in this latter case, detection is effected by way of a label which is fixed to this ligand.
Radioactive or cold labels can be used.
A large number of labelling techniques are described, for example by SAMBROOK et al., [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)]. In the case of radioactive labels, these methods include, for example, labelling synthetic oligonucleotides 5′ with &ggr;
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P-ATP using the enzyme polynucleotide kinase, or else incorporating radioactive dCTP or dATP while labelling by nick translation.
Biotin may be mentioned, in particular, as an example of a non-radioactive label which can be incorporated chemically into synthetic oligonucleotides (Application PCT WO 89/12462); biotinylated analogues of UTP can also be incorporated into double-stranded DNAs by nick translation.
In general, biotin is detected by being bound to avidin, which is itself used as a support for attaching fluorescent molecules, enzymes or other detectable compounds.
In other non-radioactive labelling systems, haptens (dinitrophenyl group, digoxigenin) replace biotin and specific labelled antibodies are used as indicators in the system.
The sensitivity of molecular hybridization assays is an important factor which is often limiting when applying diagnostic tests in molecular biology.
The sensitivity of an assay depends directly on the labelling which is used to visualize the hybridized probe. One of the ways of improving the sensitivity of an assay is to amplify the signal by increasing the number of labels which are incorporated into the probe in order to obtain so-called “polylabelled” probes.
With this aim in mind, it has been proposed, for example, that the enzyme terminal transferase should be used, which enzyme is able to extend a single-stranded DNA chain by adding nucleotide analogues, for example biotinylated nucleotides, to its 3′ end. The main drawback of this method is that the enzyme adds an arbitrary number of nucleotides to the end of the single-stranded DNA chain. This results in a mixture of products of different lengths which in turn makes the labelling difficult to reproduce and produces heterogeneous probes which are difficult to standardize and therefore unsuitable for medical diagnosis.
COLLINS (Application EP 204 510) proposes a variant in which the enzyme terminal transferase is used to extend the probe by adding a homopolymeric (poly A) tail to it. The label then consists of a homopolymer (poly T) into which detectable molecules are incorporated. However, this system does not completely eliminate the drawbacks which result from the impossibility of controlling the action of the enzyme.
It has also been proposed to amplify the signal by using polylabelled oligonucleotide probes which are branched or else connected to each other by polynucleotide “bridges” which hybridize to their ends, as described in Applications EP 292 128 and EP 450 594, respectively, which are in the name of SEGEV.
U.S. Pat. No. 4,882,269 describes a system in which polylabelled secondary probes hybridize to several sites on a primary probe.
Application EP 0 317 077 and also Application PCT WO 92/02526 describe a system in which the secondary probes consist of polylabelled polynucleotide constructs in comb form.
These systems suffer from the drawback of requiring the synthesis of a large number of different oligonucleotides and the implementation of hybridization and/or ligation steps in order to form the final constructs. In addition, it is difficult to obtain final products having uniform characteristics and it is awkward to purify these products.
In particular, if there is a need to detect several different target sequences, it is then necessary, for each of these sequences, either to synthesize a new probe or to use an adapter, which is an oligonucleotide which hybridizes with the target sequence to be detected, on the one hand, and with the probe, on the other hand.
SUMMARY OF THE INVENTION
The object of the present invention is to propose a system for amplifying a hybridization signal, which system does not suffer from the abovementioned drawbacks and is simple to obtain, to purify and to use, in particular when simultaneously detecting several nucleic acid sequences.
With this aim in mind, the inventors had the idea of using nucleic acid molecules of a specific structure as labelled detection probes, which molecules had only previously been used as amplification products of a target sequence which were intended to be attached to a solid support and then detected by means of standard methods, for example using a probe.
Such nucleic acid molecules are described by NEWTON et al. [Nucleic Acids Res., 21, pp. 1155-1162, (1993)], as well as in Application EP 416 817, in the name of IMPERIAL CHEMICAL INDUSTRIES PLC. They are present in the form of a double-stranded domain (which is a copy of the target sequence) which domain is provided, at at least one of its ends, with a single-stranded tail which enables the molecule to be attached to a solid support or to be visualized using a labelled probe which is complementary to the said tail. The double-stranded domain is separated from the single-stranded tail by a region which comprises a stop synthon.
These nucleic acid molecules are obtained by the polymerase chain amplification (PCR amplification) of the target sequence using primers which comprise a nucleotide sequence which is able to hybridize with the said target sequence and a polynucleotide tail, with the nucleotide sequence and the polynucleotide tail being separated by a stop synthon.
A “synthon” is a molecule (nucleotide analogue or other molecule) which is able to be incorporated into a synthetic polynucleotide. A “stop synthon” is a synthon which additionally possesses the property of causing the polymerase to stop when the latter encounters a synthon on the template strand during a reaction for copying or elongating a polynucleotide.
When a primer which contains a stop synthon is used for amplifying a target sequence in a polymerase chain amplification reaction, the polynucleotide tail which is located beyond the stop synthon is not copied and the final amplification product is therefore present in the form of a double-stranded copy of the target sequence, which copy is provided with a single-stranded tail.
The present invention relates to the use of nucleic acid molecules possessing the structure: single-stranded region/stop synthon/double-stranded region, as described above, as probes for detecting a target sequence. More precisely, the present invention relates to a process for detecting at least one target nucleic acid sequence, characterized in that the said target sequence is brought into contact with at least one detection probe which consists of a nucleic acid molecule which consists of:
an attachment domain A, which consists of a single-stranded polynucleotide;
an intermediate domain B, which consists of at least one stop synthon; and
a visualization domain C, which consists of a double-stranded polynucleotide into wh
Bazin Herve
Sauvaigo Sylvie
Teoule Robert
Alston & Bird LLP
Cis Bio International
Lu Frank
Sisson Bradley L.
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