Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1995-04-24
1998-12-15
Arthur, Lisa B.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 912, 536 2433, 935 77, 935 78, C12P 1934, C07H 2104
Patent
active
058495479
DESCRIPTION:
BRIEF SUMMARY
This application was filed under 35U.S.C.371 from PCT/FRA4/00935 filed Jul. 26, 1994.
FIELD OF THE INVENTION
The present invention refers to the methods, reagents and kits for the amplification of target nucleic acid sequences. In particular, the present invention consists in a method for the amplification of nucleic acids by transcription, using displacement, and the detection of the amplification products obtained by this reaction.
DESCRIPTION OF THE RELATED ARTS
It is often necessary, in technologies relating to nucleic acids and to genetic material, to determine if a gene, a gene portion or a nucleotide sequence is present in a living organism, a cellular extract of this organism or a biological sample. Since any gene or gene portion is a specific sequence of nucleotide bases forming all or part of a nucleotide molecule, it is possible to directly search for the presence of a specific nucleotide sequence in a sample containing polynucleotides.
The usefulness of the search for specific nucleotide sequences is immense, especially for the detection of pathogenic organisms, the determination of the presence of alleles, the detection of the presence of lesions in a host genome and the detection of the presence of a specific mRNA or of the modification of a cellular host. Genetic diseases such as Huntington's disease, Duchenne's myopathy, phenylketonuria and .beta.-thalassemia can be diagnosed by analysing DNA from the individual. Furthermore, the diagnosis or the identification of viruses, viroids, bacteria, fungi, protozoa or some other form of plant or animal life can be carried out by hybridization experiments with nucleic probes.
Various types of methods for the detection of nucleic acids are described in the literature. These methods, and particularly those which require the detection of polynucleotides, are based on the purine-pyrimidine pairing properties of the complementary strands of nucleic acids in the DNA-DNA, DNA-RNA and RNA--RNA complexes. This pairing process occurs by the establishment of hydrogen bonds between the adenosine-thymine (A-T) and guanosine-cytosine (G-C) bases of the double-stranded DNA; adenosine-uracil (A-U) base pairs can also be formed by hydrogen bonding in the DNA-RNA or RNA--RNA duplexes. The pairing of nucleic acid strands for the determination of the presence or of the absence of a given nucleic acid molecule is commonly called "nucleic acid hybridization" or simply "hybridization".
In the few examples mentioned above, after identifying a sequence specific for an organism or for a disease, it is advisable to extract the nucleic acids from a sample, and to determine if this sequence is present. Numerous detection methods have been developed for this purpose.
While it is vital that one or more sequences specific for a disease or for an organism are identified, the nature of these sequences and the manner in which they were identified is of no particular importance for the implementation of the present invention. The most direct method for detecting the presence of a target sequence in a nucleic acid sample is to obtain a "probe" whose sequence is sufficiently complementary to a portion of the target nucleic acid to hybridize to the latter. The probe thus synthesized can be applied in a sample containing nucleic acids, and if the target sequence is present, the probe will hybridize in order to form a reaction product. In the absence of target sequence and by avoiding any non-specific hybridization phenomenon, no reaction product will be formed. If the synthesized probe is coupled to a detectable marker, the reaction product can be detected by measuring the quantity of marker present. Southern blotting (Southern E. M., J. Mol. Biol., 98, 503 (1975)) or sandwich hybridization (Dunn A. R., Hassel J.A., Cell, 12, 23 (1977)) constitute examples where these methods are used.
The principal difficulty of this approach is, however, that it is not directly applicable to cases where the copy number of the target sequence present in a sample is low (that is to say less than 10.
REFERENCES:
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patent: 5422252 (1995-06-01), Walker et al.
patent: 5437990 (1995-08-01), Burg et al.
patent: 5480784 (1996-01-01), Kacian et al.
Gingeras, T. et al., "Unique Features of the Self-Sustained Sequence Replication (3SR) Reaction in Vitro Amplification of Nucleic Acids," Annales De Biologie Clinique, vol. 48, pp. 498-501 1990, Paris, FR.
Cleuziat Philippe
Guillou-Bonnici Francoise
Levasseur Pierre
Mallet Francois
Arthur Lisa B.
Bio Merieux
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