Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1995-08-21
1999-10-19
Houtteman, Scott W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C07H 2104
Patent
active
059691280
DESCRIPTION:
BRIEF SUMMARY
This invention relates to non-radioactive nucleic acid probes, and to chemical compounds useful in the synthesis of the said probes. It is becoming increasingly common to use nucleic acid probes for the detection and diagnosis of hereditary genetic diseases, oncogenes, and diseases caused by viruses, bacteria or parasites, and they are being employed in clinical and veterinary fields and in the food-growing industry. The probes concerned are generally single-stranded DNA or RNA sequences which are capable, under certain experimental conditions, of regaining their complementary sequences and of forming hybrids with them so as to produce stable duplexes.
Originally, the method employed to detect DNA-DNA or DNA-RNA hybrids called for the use of radioactive marking: the probe is marked with a radioisotope, usually a .sup.32 P radioisotope, and detection following hybridization entails counting or autoradiography.
In view of the drawbacks associated with the use of radioisotopes (period of decay, cost and security), there is a growing trend towards the use of "cold" probes (which do not contain a radioactive element). Cold probes employ detection techniques principally utilizing enzymatic systems which give rise to the production of a colour in the presence of a substrate, and, in the most recent systems, to the production of a light (fluorescence, chemiluminescence or bioluminescence).
As far as the marking of such probes is concerned, the techniques published to date may be broken down into two major categories:
1. The first consists in splicing the probe to a directly detectable element, for example by covalent splicing of an enzyme to the probe (alkaline phosphatase or peroxydase).
2. The second category consists in the indirect detection of the hybrid. This involves the use of intermediate substances which recognize the units attaching to the probe. These are chiefly systems based on very vigorous interaction between biotin and avidin or streptavidin, where avidin or streptavidin are paired with an enzyme. This approach requires one or more biotins to be incorporated in the nucleic probe.
Techniques based on the use of cold probes, whether involving direct or indirect detection, therefore necessitate chemical modifications to the oligonucleotide chain, at the said probes, in order to enable it to be marked.
Oligodeoxyribonucleotides bearing chemical groups such as primary amines or thiols which enable splicing with varied reagents have been described in the literature. The introduction of these groups may take place either on one or more of the bases, or at one of the 5' or 3' ends of the oligodeoxyribonucleotide. Chemical modifications at the bases of the chain have the disadvantage of interfering with the pairings of the bases when the oligonucleotide is made to form a hybrid with homologous sequences. To this end it is more judicious to introduce functional groups at 5' or at 3', provided that the technique proposed allows the detectable portion to be sufficiently removed from the oligonucleotide portion of the probe thus prepared.
It is therefore an object of this invention to obtain nucleic acid probes that: the lowest possible detection threshold; and manual synthesis of nucleic acids, notably on a solid support.
Accordingly, the present invention concerns nucleic acid probes for detecting a DNA or RNA molecule, comprising DNA or RNA nucleic acid sequence, according to the type of molecule to be detected; and direct or indirect attachment of one or more detection units or marking elements M detectable in a non-isotopic manner by producing colour or light, characterized in that portion b) is constituted by a chain of phosphate units interspersed with alkyl units, viz: no particular functionality; varied reagents in order to achieve direct or indirect detection; units b2) are linked to portion a) or sequence S via units b1).
In accordance with the invention, sequence S is linked at its 5' and/or 3' end to one or more marking elements M.
The molecular structure having the X chemical groups enabling dire
REFERENCES:
patent: 4743535 (1988-05-01), Carico
patent: 4910300 (1990-03-01), Urdea
patent: 5124246 (1992-06-01), Urdea et al.
N.D. Sinha et al., "Polymer support olgionucleotide systhesis XVIII.sup.121 ", Nucleic Acids Research, vol. 12, No. 11, 1984.
Bollen Alex
De Vos Marie-Joelle
Houtteman Scott W.
La Region Wallone
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