WO2002002803A2

WO2002002803A2 - Method for formulating molecular probes

Info

Publication number: WO2002002803A2
Application number: PCT/CH2001/000381
Authority: WO
Inventors: Werner Alex Hafner; Pierre-Alain Menoud
Original assignee: Werner Alex Hafner; Menoud Pierre Alain
Priority date: 2000-07-03
Filing date: 2001-06-18
Publication date: 2002-01-10
Also published as: WO2002002803A3; AU2001263708A1

Abstract

The invention concerns a method for selecting and formulating non-contagious molecular probes used for detecting nucleic acids. It concerns a contamination-free genetic diagnostic method for direct quantitative and qualitative analysis and gene typing of infectious agents whether pathogenic or not without prior RNA or DNA extraction. The probes produced are very specific, highly sensitive and enable direct detection of nucleic acids in sections of living or immobilised, frozen or fixed and/or coated prokaryotic and eukaryotic organism tissues and in particular retroviral particles circulating in the biological fluids or present in the cells.

Claims

CLAIMS We claim:

1. A process for the formulation of molecular probes which takes into account the forms of presentation of acellular and cellular organisms during their life cycle, where the forms of presentation are dependent on the different chemical, biological and physical states of the organisms.

2. A process for the formulation of molecular probes which takes into account the criimical structures defined by regions rich in guanine and cytosine whose concentration is between 20% and 70% over a sequence length between 10 and 1000 bases and which are insensitive to degradation by enzymes or ionizing rays and / or which are protective regions of other sequences and structures composed of nucleic acids which have a free palindromic binding energy of between -300 kCal / mole and -5 kCal /mole.

3. A process for the formulation of molecular probes consisting of ribonucleic or deoxyribonucleic acids or of derivatives of synthetic nucleic acids intended for the hybridization of nucleic acids of prokaryotic, eukaryotic and viral organisms, complete or fragmented, according to which the probes recognize one or more endodogmatic sites defining the modes of biological presentation; endodogmatic sites are nucleic acid sequences made up of at least one consensus sequence specific to a type, a genotype, a species or a family and conserved during generations of individuals and necessary for the determination of adjacent sequences with a length between 3 to 100 bases.

4. A process for the formulation of molecular probes consisting of ribonucleic or deoxyribonucleic acids or of derivatives of synthetic nucleic acids intended for the hybridization of nucleic acids of prokaryotic, eukaryotic and viral organisms, complete or fragmented, according to which the probes recognize exodogmatic sites defining the modes of biological presentation are sequences of nucleic acids conserved or not during generations of individuals and necessary or not for their reproduction or their functionalities; these exodogmatic sites themselves consist of at least one consensus sequence specific to a type, a genotype, a species or a family.

5. A process for the formulation of molecular probes consisting of ribonucleic or deoxyribonucleic acids or of derivatives of synthetic nucleic acids intended for the hybridization of nucleic acids of prokaryotic, eukaryotic and viral organisms, complete or fragmented, according to which the properties Physics are defined by three-dimensional structures that integrate the chemical and biological properties of specific nucleic acid sequences.

6. The use of the chemical properties, the biological characteristics and the physical properties of the nucleic acids as described in the implementation of the invention for the formulation, the manufacture and the production, by natural or artificial methods, of antibodies or fragments of antibodies directed against nucleic acids, against endodogmatic sites and / or their adjacent sequences consisting of 3 to 500 bases, against a series of natural amino acids or

1 7 synthetics translated from nucleic acid sequences including all or part of at least one endodogmatic site.

7. The use of the chemical properties, the biological characteristics and the physical properties of the nucleic acids as described in carrying out the invention for the formulation, the manufacture and the production, by natural or artificial methods, of antibodies or antibody fragments directed against exodogmatic sites and / or their adjacent sequences consisting of 3 to 500 bases, against a series of natural or synthetic amino acids translated from the nucleic acid sequences including all or part of at least an exodogmatic site.

8. A method in which molecular probes are formulated so that they are partly in a loop of the hairpin of the target sequence and of which the nucleic acid sequence of the stem contains 80% or less of bases G and C and whose three-dimensional spatial structure means that on either side of the sequence of molecular probes an RNA or a single-stranded DNA exists and whose spatiality is used for the recognition of nucleic acid sequences d '' a length of 10 to 10,000 bases and comprising at least one endodogmatic site and consisting of at least one consensus sequence specific to a type, to a genotype, to a species or to a family.

9. A method according to which molecular probes are formulated so that they are partly in a loop of the hairpin of the target sequence and of which the nucleic acid sequence of the stem contains 80% or less of bases G and C and whose three-dimensional spatial structure means that on either side of the sequence of molecular probes an RNA or a single-stranded DNA exists and whose spatiality is used for the recognition of nucleic acid sequences d '' a length of 10 to 10,000 bases and comprising at least one exodogmatic site and consisting of at least one consensus sequence specific to a type, to a genotype, to a species or to a family.

10. A method by which molecular probes are labeled with fluorescent molecules or with biotin or with electron dense particles or with haptens or with enzymes or labeled with at least one radioactive substance.

11. A method according to claims 1 to 10, for the formulation of molecular probes intended for the detection of RNA or DNA of viral origin, including oncogenes, or bacteria recognized or not by the human, animal or vegetal.

12. A method according to claims 1 to 10, in which molecular probes are formulated for in situ hybridization or detection of nucleic acids.

13. A method according to claims 1 to 5 in which primers are formulated for use in a polymerase chain reaction (PCR ^® ).

14. A method according to claims 1 to 10, according to which there results an automation of the method for detecting viral or bacterial nucleic acids or other microorganisms, with or without prior extraction of nucleic acids and from biological fluids such as plasma , serum, blood components, semen, urine as well as samples including cells, cellular components and human, animal or plant tissue.

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15. A method according to claims 1 to 9, according to which molecular probes are formulated for the recognition of endodogmatic sites or of adjacent sequences surrounding at least one endodogmatic site by polymerase chain reaction, by detection with antibodies or ribozymes, by nucleic acid hybridization with or without prior digestion with a specific or non-specific protease of living or dead tissues, cells, biological fluids such as plasma, serum, blood components, sperm, urine.

16. A method according to claims 1 to 9, according to which molecular probes or antibodies or antibody fragments are formulated for blocking the reproduction of pathogens or living organisms and / or their use for therapeutic purposes .

17. A method according to claims 1 to 9, according to which ribozymes are formulated for the digestion or for the fragmentation of natural or synthetic nucleic acids.

18. A method according to claims 8 and 9, according to which molecular probes are formulated for hybridization and / or detection of nucleic acids in order to determine the presence and or the genotype by means of microplates or solid supports. miniaturized used in nanotechnology on which are fixed synthetic or natural nucleic acids.

A computer program for the choice and the formulation of specific molecular probes and / or for the detection of nucleic acids and / or for a use according to claims 1 to 18, integrating the functionality of a spatial conformation of the nucleic acids (physical properties ) whose spatiality is used to recognize a specific energy or endodogmatic sites and or sequences surrounding at least one endodogmatic site (biological properties) the thermodynamics of the connections between nucleotides and the parameters of a secondary structure (chemical properties).

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