Methods and compositions for detection or quantification of nucleic acid species

• US 20030108897A1
• Filed: 07/01/2002
• Published: 06/12/2003
• Est. Priority Date: 01/16/1997
• Status: Abandoned Application

• Current Assignee: Radoje T. Drmanac
• Original Assignee: Radoje T. Drmanac
• Inventors: Drmanac, Radoje T.
• Application Number:
  US10/187,251
• Publication Number:
  US 20030108897A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• 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 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• 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 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• 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: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to oligonucleotide probes attached to discrete particles wherein the particles can be grouped into a plurality of sets based on a physical property. A different probe is attached to the discrete particles of each set, and the identity of the probe is determined by identifying the discrete particles from their physical property. The physical property includes any that can be used to differentiate the discrete particles, and includes, for example, size, flourescence, radioactivity, electromagnetic charge, or absorbance, or label(s) may be attached to the particle such as a dye, a radionuclide, or an EML. In a preferred embodiment, discrete particles are separated by a flow cytometer which detects the size, charge, flourescence, or absorbance of the particle. The invention also relates to methods using the probes complexed with the discrete particles to analyze target nucleic acids.

Claims

1. A method for analyzing a target nucleic acid, comprising the steps of:

contacting the target nucleic acid with a plurality of oligonucleotide probes, wherein the probes are complexed with a plurality of different discrete particles that can be discriminated from one another based on a physical property and wherein a different probe is complexed with each type of discrete particle;

detecting those probes that are complementary to the target nucleic acid; and

analyzing the target nucleic acid from a set of complementary probes.

2. The method of claim 1, further comprising the step of separating the discrete particles into fractions, wherein the discrete particles are separated on the basis of the physical property.

3. The method of claim 2, wherein the set of complementary probes has at least two probes which overlap.

4. The method of claim 2, wherein a sequence of the target nucleic acid is compiled in the analyzing step.

5. The method of claim 2, wherein the complementary probes are identified by the physical property of the discrete particle.

6. The method of claim 2, wherein wherein the physical property is associated with a molecule selected from the group consisting of a dye, a radionucleotide, an EML, and a flourescent molecule.

7. The method of claim 2, wherein the physical property is selected from the group comprising a size, a charge, an absorbance, and a weight.

8. The method of claim 7, wherein the physical property is associated with an intensity of the physical property.

9. The method of claim 7, wherein the physical property is associated with a plurality of different molecules.

10. The method of claim 2, wherein an informative portion of the probes is shorter than the probes full length.

11. The method of claim 2, wherein the target nucleic acid contains a label, and the complementary probes are detected by the label on the target nucleic acid.

12. The method of claim 1, wherein the detecting step is performed on individual discrete particles by passing the individual particles past a detector.

13. The method of claim 12, wherein the set of complementary probes has at least two probes which overlap.

14. The method of claim 12, wherein a sequence of the target nucleic acid is compiled in the analyzing step.

15. The method of claim 12, wherein the complementary probes are identified by the physical property of the discrete particle.

16. The method of claim 12, wherein wherein the physical property is associated with a molecule selected from the group consisting of a dye, a radionucleotide, an EML, and a flourescent molecule.

17. The method of claim 12, wherein the physical property is selected from the group comprising a size, a charge, an absorbance, and a weight.

18. The method of claim 17, wherein the physical property is associated with an intensity of the physical property.

19. The method of claim 17, wherein the physical property is associated with a plurality of different molecules.

20. The method of claim 12, wherein an informative portion of the probes is shorter than the probes full length.

21. The method of claim 12, wherein the target nucleic acid contains a label, and the complementary probes are detected by the label on the target nucleic acid.

22. The method of claim 12, further comprising the step of contacting the target nucleic acid with a plurality of free oligonucleotides, and

covalently joining a complementary free probe, bound at a site in the target nucleic acid, to a complementary probe complexed to the discrete particle which is bound to a site on the target nucleic acid that is adjacent to the site on which the free probe is bound, and

wherein the detecting step identifies the free probe which is covalently joined to the probe of the discrete particle.

23. The method of claim 22, wherein the set of complementary, covalently joined probes has at least two covalently joined probes which overlap.

24. The method of claim 22, wherein a sequence of the target nucleic acid is compiled in the analyzing step.

25. The method of claim 22, wherein the probes complexed to the discrete particle are identified by the physical property of the discrete particle.

26. The method of claim 22, further comprising the step of separating the discrete particles into fractions, wherein the discrete particles are separated on the basis of the physical property.

27. The method of claim 26, wherein the discrete particles are separated into fractions using a flow cytometer.

28. The method of claim 22, wherein the physical property is associated with a molecule selected from the group consisting of a dye, a radionucleotide, an EML, and a flourescent molecule.

29. The method of claim 22, wherein the physical property property is selected from the group comprising a size, a charge, an absorbance, and a weight.

30. The method of claim 29, wherein the physical property is associated with an intensity of the physical property.

31. The method of claim 29, wherein the physical property is associated with a plurality of different molecules.

32. The method of claim 22, wherein an informative portion of the free probes is shorter than the probes full length.

33. The method of claim 22, wherein an informative portion of the probes complexed to the discrete particles is shorter than the probes full length.

34. The method of claim 22, wherein an informative portion of the free probes and an informative portion of the probes complexed to the discrete particles are shorter than the probes full length.