Detection and quantification process for more than one nucleic acid in library
This invention provides novel compositions and processes for analyte detection, quantification and amplification. Nucleic acid arrays and libraries of analytes are usefully incorporated into such compositions and processes. Universal detection elements, signaling entities and the like are employed to detect and if necessary or desirable, to quantify analytes. Amplification of target analytes are also provided by the compositions and processes of this invention.
1. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent heteropolymeric universal detection targets (UDT) to a nucleic acid, wherein said attaching means is selected from blunt-end ligation with DNA ligase, ligation of single-stranded ends with RNA ligase or a combination thereof;
(iv) a universal detection element (UDE) which generates a signal directly or indirectly;
b) attaching said non-inherent UDTs to said library of nucleic acid analytes;
c) hybridizing said library of nucleic acid analytes from step b) with said array of fixed or immobilized nucleic acids to form hybrids if said nucleic acids of interest are present;
d) contacting said UDE with said non-inherent UDTs to form a complex bound to said array; and
e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from any UDE bound to said array.
2. The process of claim 1 , wherein said nucleic acid array comprises DNA, RNA, DNA analogs, RNA analogs, or a combination thereof.
3. The process of claim 2 , wherein said nucleic acid array comprises analogs comprising PNA.
4. The process of claims 2 or 3 , wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.
5. The process of claim 1 , wherein said solid support is porous or non-porous.
6. The process of claim 5 , wherein said solid support is porous and comprises polyacrylamide or agarose.
7. The process of claim 5 , wherein said solid support is non-porous and comprises glass or plastic.
8. The process of claim 1 , wherein said solid support is transparent, translucent, opaque or reflective.
9. The process of claim 1 , wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.
10. The process of claim 9 , wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.
11. The process of claim 1 , wherein said library of analytes is derived from a biological source comprising an organ, a tissue, a cell, or a combination thereof.
12. The process of claim 1 , wherein said analytes comprise genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA, or a combination thereof.
13. The process of claim 1 , wherein said UDE comprises a nucleic acid, a nucleic acid analog, or a modified form thereof.
14. The process of claim 13 , wherein said UDE comprises a nucleic acid analog, wherein the nucleic acid analog comprises PNA.
15. The process of claim 1 , wherein said UDE generates a signal directly by a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound, or a combination of any of the foregoing.
16. The process of claim 1 , wherein said UDE generates a signal indirectly by an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme, or a combination of any of the foregoing.
17. The process of claim 16 , wherein said UDE generates a signal by an enzyme that catalyzes a reaction comprising a fluorogenic reaction, a chromogenic reaction a chemiluminescent reaction, or a combination thereof.
18. The process of claim 1 , comprising one or more washing steps.
19. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent heteropolymeric universal detection targets (UDT) to a nucleic acid, wherein said attaching means is selected from blunt-end ligation with DNA ligase, ligation of single-stranded ends with RNA ligase or a combination thereof;
(iv) a universal detection element (UDE) which generates a signal directly or indirectly;
b) attaching said non-inherent UDTs to said library of nucleic acid analytes;
c) contacting said UDE with said non-inherent UDTs in said library of nucleic acid analytes from step b) to form one or more complexes;
d) hybridizing said library of nucleic acid analytes from step c) with said array of nucleic acids to form hybrids if said nucleic acids of interest are present; and
e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from any UDE bound to said array.
20. The process of claim 19 , wherein said nucleic acid array comprises DNA, RNA, DNA analogs, RNA analogs, or combinations thereof.
21. The process of claim 20 , wherein said nucleic acid array comprises analogs comprising PNA.
22. The process of claims 20 or 21 , wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.
23. The process of claim 19 , wherein said solid support is porous or non-porous.
24. The process of claim 23 , wherein said solid support is porous and comprises polyacrylamide or agarose.
25. The process of claim 23 , wherein said solid support is non-porous and comprises glass or plastic.
26. The process of claim 19 , wherein said solid support is transparent, translucent, opaque or reflective.
27. The process of claim 19 , wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.
28. The process of claim 27 , wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.
29. The process of claim 19 , wherein said library of analytes is derived from a biological source comprising an organ, a tissue, a cell, or a combination thereof.
30. The process of claim 19 , wherein said analytes comprise genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA, or a combination thereof.
31. The process of claim 19 , wherein said UDE comprises a nucleic acid, a nucleic acid analog, or a modified form thereof.
32. The process of claim 31 , wherein said UDE comprises a nucleic acid analog comprising PNA.
33. The process of claim 19 , wherein said UDE generates a signal directly by a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound, or a combination of any of the foregoing.
34. The process of claim 19 , wherein said UDE generates a signal indirectly by an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme, or a combination of any of the foregoing.
35. The process of claim 34 , wherein said UDE generates a signal by an enzyme that catalyzes a reaction comprising a fluorogenic reaction, a chromogenic reaction a chemiluminescent reaction, or a combination thereof.
36. The process of claim 19 , comprising one or more washing steps.
37. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and
(iii) a universal detection element (UDE) which binds to a domain formed by nucleic acid hybrids for complex formation and generates a signal directly or indirectly, wherein said UDE is selected from a protein, an intercalator, and a combination thereof;
b) hybridizing said library of nucleic acid analytes with said array of nucleic acids to form hybrids if said nucleic acids of interest are present, wherein said formed hybrids generate a domain for complex formation;
c) contacting said UDE with said hybrids to form a complex bound to said array; and
d) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from any UDE bound to said array.
38. The process of claim 37 , wherein said nucleic acid array comprises DNA, RNA, DNA analogs, RNA analogs, or combinations thereof.
39. The process of claim 38 , wherein said nucleic acid array comprises analogs comprising PNA.
40. The process of claims 38 or 39 , wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.
41. The process of claim 37 , wherein said solid support is porous or non-porous.
42. The process of claim 41 , wherein said solid support is porous and comprises polyacrylamide or agarose.
43. The process of claim 41 , wherein said solid support is non-porous and comprises glass or plastic.
44. The process of claim 37 , wherein said solid support is transparent, translucent, opaque or reflective.
45. The process of claim 37 , wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.
46. The process of claim 45 , wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.
47. The process of claim 37 , wherein said library of analytes is derived from a biological source comprising an organ, a tissue, a cell, or a combination thereof.
48. The process of claim 37 , wherein said analytes comprise genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA, or a combination thereof.
49. The process of claim 37 , wherein said domain for complex formation comprises DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids, RNA-PNA hybrids, or a combination thereof.
50. The process of claim 37 , wherein when said UDE is a protein, the protein is a nucleic acid binding protein which binds to double-stranded nucleic acids more than single-stranded nucleic acids.
51. The process of claim 50 , wherein when said UDE is a nucleic acid binding protein the nucleic acid binding protein is an antibody.
52. The process of claim 51 , wherein said antibody is specific for nucleic acid hybrids comprising DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids, RNA-PNA hybrids, and a combination thereof.
53. The process of claim 37 , wherein when said UDE is an intercalator, said UDE is selected from ethidium bromide, diethidium bromide, acridine orange, SYBR Green, or a combination thereof.
54. The process of claim 37 , wherein said UDE generates a signal directly by a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound, or a combination of any of the foregoing.
55. The process of claim 37 , wherein said UDE generates a signal indirectly by an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme, or a combination of any of the foregoing.
56. The process of claim 55 , wherein said UDE generates a signal by an enzyme that catalyzes a reaction comprising a fluorogenic reaction, a chromogenic reaction a chemiluminescent reaction, or a combination thereof.
57. The process of claim 37 , further comprising one or more washing steps.