Process for aligning macromolecules by passage of a meniscus and applications

• US 20030175779A1
• Filed: 02/13/2003
• Published: 09/18/2003
• Est. Priority Date: 02/11/1994
• Status: Active Grant

• Current Assignee: Institut Pasteur and Centre National de La Recherche Scientifique
• Original Assignee: Institut Pasteur and Centre National de La Recherche Scientifique
• Inventors: Bensimon, Aaron, Bensimon, David, Heslot, Francois
• Granted Patent: US 7,122,647 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00628 Ionic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/97 : Test strip or test slide
• CPC Class Codes: Y10S 530/81 : Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
• CPC Class Codes: Y10S 530/811 : Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/145555 : Hetero-N

Abstract

The subject of the present invention is a process for aligning a macromolecule (macromolecules) on the surface S of a support, characterized in that the triple line S/A/B (meniscus) resulting from the contact between a solvent A and the surface S and a medium B is caused to move on the said surface S, the said macromolecules having a part, especially an end, anchored on the surface S, the other part, especially the other end, being in solution in the solvent A.

The subject of the present invention is also a process for detecting, measuring the intramolecular distance of, separating and/or assaying a macromolecule in a sample in which a process of alignment according to the invention is used.

Claims

1. Process for aligning a macromolecule (macromolecules) on the surface S of a support, characterized in that the triple line S/A/B (meniscus) resulting from the contact between a solvent A and the surface S and a medium B is caused to move on the said surface S, the said macromolecules having a part, especially an end, anchored on the surface S, the other part, especially the other end, being in solution in the solvent A.

2. Process according to claim 1, characterized in that the movement of the meniscus is achieved by evaporation of the solvent A.

3. Process according to claim 1, characterized in that the movement of the meniscus is achieved by relative movement of the A/B interface relative to the surface S.

4. Process according to claim 3, characterized in that in order to move the meniscus, the surface S is removed from the solvent A or the solvent A is removed from the surface S

5. Process according to one of claims 1 to 4, characterized in that the meniscus is a water/air meniscus.

6. Process according to one of claims 1 to 5, characterized in that the support consists, at least at the surface, of an organic or inorganic polymer, a metal, a metal oxide or sulfide, a semiconductor element such as silicon or an oxide of a semiconductor element, or one of the combinations thereof.

7. Process according to one of claims 1 to 6, characterized in that the support consists, at least at the surface, of glass, surface-oxidized silicon, gold, graphite, molybdenum sulfide or mica.

8. Process according to one of claims 1 to 7, characterized in that the support is in the form of a plate, bead, fiber or particle system.

9. Process according to one of claims 1 to 8, characterized in that the solvent A in which the macromolecules to be aligned are in solution is placed between two supports, of which one at least corresponds to the said support of surface S, and the meniscus is moved by evaporation.

10. Process according to one of claims 1 to 9, characterized in that the anchoring of the macromolecule is carried out by physicochemical interaction, especially by adsorption or by covalent linkage, either directly between the surface and the macromolecule, or indirectly between the surface and another molecule recognizing and/or interacting with the said macromolecule.

11. Process according to one of claims 1 to 10, characterized in that a support is used which has at the surface an exposed reactive group having an affinity for the said macromolecule or a molecule with biological activity capable of recognizing the said macromolecule.

12. Process according to one of claims 1 to 11, characterized in that the surface is coated with a group chosen from the vinyl, amine, carboxyl, aldehyde or hydroxyl groups.

13. Process according to either of claims 10 and 12, characterized in that the surface contains:

on a support, a substantially monomolecular layer of an organic compound of elongated structure having at least;

an attachment group having an affinity for the support, and

an exposed group having no or little affinity for the said support and the said attachment group under attachment conditions, but optionally having, after chemical modification following the attachment, an affinity for the said macromolecule or molecule with biological activity.

14. Process according to one of claims 1 to 13, characterized in that the anchoring of a part of a macromolecule is carried out by adsorption on a surface, by bringing the said macromolecule into contact with the said surface in a determined region of pH or ionic content of the medium or by applying a determined electric voltage on the anchoring surface.

15. Process according to claims 1 to 14, characterized in that the pH for carrying out the anchoring is chosen in a range between a pH favoring a state of complete adsorption and a pH favoring absence of adsorption.

16. Process according to one of claims 1 to 15, in which the anchoring of a nucleic acid or of a protein is carried out by adsorption on a surface having groups containing ethylenic double bonds or amine groups, by bringing the nucleic acid or the protein into contact with the surface in a determined region of pH or ionic content of the medium.

17. Process according to claim 16, characterized in that the anchoring of nonfunctionalized DNA is carried out by adsorption on surfaces coated with molecules ending with a vinyl or amine group.

18. Process according to claim 17, in which the anchoring of the DNA is carried out by its end onto a surface having groups with ethylenic double bonds, by bringing the DNA into contact with the surface at a pH of less than 8.

19. Process according to claim 18, characterized in that the reaction is carried out at a pH of between 5 and 6, and is then stopped at pH 8.

20. Process according to claim 17, characterized in that the anchoring of DNA is carried out by its end onto a surface coated with polylysine or a silane group ending with an amine group.

21. Process according to claim 17, characterized in that the anchoring of the DNA is carried out by its end onto a surface coated with an amine group by bringing the DNA into contact with the surface at a pH of between 8 and 10.

22. Process according to claim 17, characterized in that the anchoring of DNA is carried out by its end onto a glass surface treated beforehand in an acid bath, by bringing the DNA into contact with the said surface at a pH of between 5 and 8.

23. Surface with aligned macromolecule(s) obtained by the process according to one of claims 1 to 22.

24. Process for detecting, separating and/or assaying a macromolecule in a sample, characterized in that a process for alignment according to one of claims 1 to 23 is used in which a molecule with biological activity capable of recognizing the said sample macromolecule becomes attached to the surface S, and in that the detection, separation or assay are carried out using a reagent, fluorescent or otherwise, which detects the presence of the attached molecule or the said macromolecule.

25. Process according to one of claims 1 to 24, characterized in that the said macromolecule and molecule with biological activity are chosen from proteins, nucleic acids, lipids, polysaccharides and derivatives thereof.

26. Process according to claim 24, characterized in that the said macromolecule and molecule with biological activity are chosen from antibodies, antigens, DNAs, RNAs, ligands or their receptors as well as derivatives thereof.

27. Process according to claims 24 to 26, characterized in that the attached DNA contains the sequence complementary to a DNA sequence to be isolated from a sample.

28. Process according to claims 24 to 26, characterized in that the attached protein is capable of specifically recognizing and attaching a protein to be isolated from a sample.

29. Process according to claims 24 to 26, characterized in that the said molecule with biological activity is chosen from biotin, avidin, streptavidin, derivatives thereof or an antigen-antibody system.

30. Process according to claims 24 to 26, characterized in that the surface exhibits low fluorescence and in that the reagent is fluorescent.

31. Process according to claims 24 to 26, characterized in that the reagent consists of beads.

32. Process according to one of claims 24 to 26, characterized in that the detection is performed by optical or near field microscopy.

33. Process according to one of claims 24 to 26, characterized in that the product of a reaction between the molecule with biological activity and the macromolecule of the sample is subjected to stress in order to destroy the mismatches before the detection.

34. Process for detecting a macromolecule consisting of a DNA sequence or a protein in a sample, according to one of claims 24 to 33, characterized in that:

the sample corresponding to solvent A, in which the said macromolecule is in solution, is brought into contact with the surface of the support under conditions for forming a DNA/DNA, DNA/RNA hybrid or for forming the protein/protein reaction product,

the hybrid or a macromolecule for labeling the hybrid or the reaction product being anchored in one part, the remainder being in solution, it is stretched by the movement of the meniscus created by the contact between the solvent

and the surface in order to orient the hybrids or the said labeling macromolecules and the measurement or the observation of the hybrids or of the said labeling macromolecules thus orientated is carried out.

35. Process according to one of claims 24 to 34, characterized in that the attached DNA and the sample DNA are “

colored”

differently and, after stretching, the position of the complementary sequence relative to the end of the sample DNA is measured.

36. Process according to one of claims 24 to 35, characterized in that an ELISA or FISH detection method is used.

37. Process according to one of claims 24 to 36, characterized in that the sample is the product or the substrate of an enzymatic amplification of nucleic acid.

38. Process according to one of claims 24 to 37, characterized in that the DNA is stretched, then denatured and hybridized with specific probes in order to determine the position or the size of one or more determined DNA sequences.

42. Process according to one of claims 34 to 37, characterized in that the DNA is stretched, then denatured and hybridized with specific probes in order to determine the presence or the absence of one or more given DNA sequences.

43. Process for diagnosing a pathology related to the presence or to the absence of a given DNA sequence specific for the said pathology, in which a process according to claim 42 is used.

44. Kit useful for carrying out a diagnostic process according to claim 43, characterized in that it contains:

a support whose surface permits the anchoring and alignment of the patient'"'"'s DNA,

probes specific for the gene involved in the sought for pathology, and

reagents for hybridization and detection of the DNA.

45. Kit useful for carrying out a diagnostic process according to claim 43, characterized in that it contains:

a support whose surface has probes specific for the gene involved in the desired pathology, the said probes being anchored and aligned on the surface;

reagents for labeling DNA, especially the patient'"'"'s DNA;

reagents for the hybridization and the detection of the DNA.

46. Process for preparing a gene from a genomic DNA, characterized in that the position of the said gene on the genomic DNA aligned by the process of one of claims 1 to 27 is identified by means of a probe specific for the said gene and the enzymatic amplification of the sequence of the said gene and/or of its flanking sequences is carried out and the amplified product is isolated.

47. DNA construct containing a gene prepared by the process of claim 46, optionally associated with a homologous or heterologous regulatory sequence.

48. Use of a gene obtained by a process according to claim 46, for preparing a DNA construct useful in a process for replacing a gene in the genome of an eukaryotic cell by targeted insertion of a foreign gene, the said foreign gene being obtained by the process of claim 46.

49. Vector useful in a process for replacing a gene in the genome of a eukaryotic cell by targeted insertion of the said foreign gene, characterized in that the said foreign gene is prepared according to the process of claim 46.

39. Process for the physical mapping of a gene on a genomic DNA in which the DNA is aligned and/or detected according to a process of one of Clains 1 to 38.

40. Process according to claim 39, characterized in that the position and the size of the desired gene on the genomic DNA are determined by hybridization with probes specific for the said gene to be mapped.

41. Kit useful for carrying out a process according to either of claims 39 and 40 comprising:

total genomic DNA from a reference host,

a support having a surface permitting anchoring and alignnent of the DNA,

probes specific for the gene to be mapped, and

reagents for hybridization and detection of the DNA.