Methods for detecting the presence, location or quantity of targets using novel dyes
The present invention provides dyes, reactive dyes and labeled reagents that may be used in the detection or quantification of desirable target molecules, such as proteins and nucleic acids. Dyes are provided that may be used free in solution where the binding of the dye to the target molecule provides signal generation. Dyes are also provided that comprise reactive groups that may be used to attach the dyes to probes that will bind to desirable target molecules. The novel dyes of the present invention have been modified by the addition of charged and polar groups to provide beneficial properties.
1. A method for detecting the presence or quantity of a target comprising the steps of:
a) providing
i) a sample where the presence or quantity of a target is desired to be detected, and
ii) a composition comprising a first portion and a second portion wherein said first portion comprises a dye and said second portion comprises a target specific moiety;
b) allowing any targets present in said sample to bind with said target specific moiety; and
c) detecting or quantifying the amount of said dye bound to any of said target in the sample, thereby detecting the presence or quantity of said target,
wherein the dye comprises
2. The method of claim 1 , wherein said target comprises an oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eukaryotic cell, eukaryotic cell component, natural drug, synthetic drug, or organic molecule.
3. The method of claim 1 , wherein said dye further comprises one or more charged groups or polar groups.
4. The method of claim 3 , wherein at least one of said one or more charged groups or polar groups is monomeric.
5. The method of claim 3 , wherein at least one of said one or more charged groups or polar groups is polymeric.
6. The method of claim 3 , wherein at least one of said one or more charged groups or polar groups is anionic.
7. The method of claim 3 , wherein at least one of said one or more charged groups or polar groups is cationic.
8. The method of claim 1 , wherein said dye further comprises one or more anionic charged groups or polar groups and one or more cationic charged groups or polar groups.
9. The method of claim 1 , wherein said target specific moiety comprises an oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin, carbohydrate, oligosaccharide, polysaccharide, natural drug, synthetic drug, or organic molecule.
10. The method of claim 1 , wherein said dye is linked to said target specific moiety through a linker arm.
11. The method of claim 10 , wherein said linker arm is attached to said target specific moiety through a bond which comprises a covalent bond, a polar bond or a coordinate bond.
12. The method of claim 9 , wherein said target specific moiety is an oligonucleotide or polynucleotide that comprises two or more dye molecules attached to separate nucleotides of said oligonucleotide or polynucleotide.
13. The method of claim 12 , wherein said two or more dye molecules comprise the same dye molecules.
14. The method of claim 12 , wherein said two or more dye molecules comprise different dye molecules.
15. The method of claim 1 , wherein said dye is part of a composite dye comprising a second dye molecule.
16. The method of claim 9 , wherein said target specific moiety is an oligonucleotide or polynucleotide that comprises unmodified nucleotides, modified nucleotides, nucleotide analogues or any combination thereof.
17. The method of claim 1 , wherein said sample i) or said composition ii) is fixed or immobilized to a solid support.
18. The method of claim 17 , further comprising carrying out one or more washing steps after said binding step b).
19. The method of claim 1 , wherein said binding step b) and said detecting step c) are carried out in the absence of washing steps.
20. The method of claim 19 , wherein said detection step c) is carried out by energy transfer.
21. A method for detecting the presence, location or quantity of a target comprising the steps of:
a) providing
i) a sample where the presence, location or quantity of a target is desired to be detected, and
ii) a dye;
b) allowing any targets present in said sample i) to bind with said dye ii); and
c) detecting the dye ii) bound to any of said target in the sample i), thereby detecting the presence, location or quantity of said target,
wherein the dye comprises
22. The method of claim 21 , wherein said target comprises an oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eukaryotic cell, eukaryotic cell component, natural drug, synthetic drug, or organic molecule.
23. The method of claim 21 , wherein said target has been bound to a solid matrix.
24. The method of claim 23 , wherein said solid matrix comprises a bead, filter, gel, cuvette, test tube, dish, microscope slide, plate, microtiter plate or microarray.
25. The method of claim 24 , wherein said target is detected in situ.
26. The method of claim 21 , wherein said detection takes place without binding of target to a solid matrix.
27. The method of claim 26 , wherein said detection takes place by a change in properties of said dye after binding takes place between said dye and said target.
28. The method of claim 27 , wherein said change is an increase or decrease in the amount of fluorescence of said dye.
29. The method of claim 23 , wherein a further step is carried out where unbound dye is removed.
30. The method of claim 1 or 21 , wherein the dye comprises
31. The method of claim 1 or 21 , wherein the dye comprises
32. The method of claim 1 or 21 , wherein the dye comprises
33. The method of claim 1 or 21 , wherein the dye comprises
34. The method of claim 1 or 21 , wherein the dye comprises
35. The method of claim 1 or 21 , wherein the dye comprises
36. The method of claim 1 or 21 , wherein the dye comprises
37. The method of claim 1 or 21 , wherein the dye comprises
38. The method of claim 1 or 21 , wherein the dye comprises
39. The method of claim 1 or 21 , wherein the dye comprises
40. The method of claim 1 or 21 , wherein the dye comprises
41. The method of claim 1 or 21 , wherein the dye comprises
42. The method of claim 1 or 21 , wherein the dye comprises
43. The method of claim 1 or 21 , wherein the dye comprises
44. The method of claim 1 or 21 , wherein the dye comprises
45. The method of claim 1 or 21 , wherein the dye comprises
46. The method of claim 1 or 21 , wherein the dye comprises
47. The method of claim 1 or 21 , wherein the dye comprises
48. The method of claim 1 or 21 , wherein the dye comprises
49. The method of claim 1 or 21 , wherein the dye comprises
50. The method of claim 1 or 21 , wherein the dye comprises
51. The method of claim 1 or 21 , wherein the dye comprises
52. The method of claim 1 or 21 , wherein the dye comprises