WO1998026098B1 - Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom - Google Patents
Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefromInfo
- Publication number
- WO1998026098B1 WO1998026098B1 PCT/US1997/023173 US9723173W WO9826098B1 WO 1998026098 B1 WO1998026098 B1 WO 1998026098B1 US 9723173 W US9723173 W US 9723173W WO 9826098 B1 WO9826098 B1 WO 9826098B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- sequence
- acid molecules
- nucleotides
- source
- Prior art date
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract 62
- 108020004707 nucleic acids Proteins 0.000 title claims abstract 56
- 239000000203 mixture Substances 0.000 title claims abstract 9
- 229920000272 Oligonucleotide Polymers 0.000 claims abstract 27
- 239000007787 solid Substances 0.000 claims abstract 20
- 229920002676 Complementary DNA Polymers 0.000 claims abstract 12
- 239000002299 complementary DNA Substances 0.000 claims abstract 12
- 229920001850 Nucleic acid sequence Polymers 0.000 claims abstract 9
- 230000002068 genetic Effects 0.000 claims abstract 8
- 210000001519 tissues Anatomy 0.000 claims abstract 4
- 239000002773 nucleotide Substances 0.000 claims 15
- 125000003729 nucleotide group Chemical group 0.000 claims 15
- 230000000295 complement Effects 0.000 claims 7
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims 5
- 230000015572 biosynthetic process Effects 0.000 claims 4
- 239000003795 chemical substances by application Substances 0.000 claims 4
- 238000005755 formation reaction Methods 0.000 claims 4
- 230000001225 therapeutic Effects 0.000 claims 4
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims 2
- 229940088592 Immunologic Factor Drugs 0.000 claims 2
- 108020004999 Messenger RNA Proteins 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 claims 2
- 201000010099 disease Diseases 0.000 claims 2
- 239000003102 growth factor Substances 0.000 claims 2
- 239000000367 immunologic factor Substances 0.000 claims 2
- 150000002611 lead compounds Chemical class 0.000 claims 2
- 229920002106 messenger RNA Polymers 0.000 claims 2
- 230000000644 propagated Effects 0.000 claims 2
- 230000001902 propagating Effects 0.000 claims 2
- 239000000126 substance Substances 0.000 claims 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 claims 1
- 241000238631 Hexapoda Species 0.000 claims 1
- 108010004729 Phycoerythrin Proteins 0.000 claims 1
- 241000700605 Viruses Species 0.000 claims 1
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine group Chemical group [C@@H]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C=NC=2C(N)=NC=NC12 OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims 1
- 230000001580 bacterial Effects 0.000 claims 1
- 238000010367 cloning Methods 0.000 claims 1
- 230000000875 corresponding Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 claims 1
- 230000002538 fungal Effects 0.000 claims 1
- 238000002372 labelling Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000035790 physiological processes and functions Effects 0.000 claims 1
- -1 small molecule compound Chemical class 0.000 claims 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims 1
- 239000011324 bead Substances 0.000 abstract 3
- 238000009396 hybridization Methods 0.000 abstract 2
- 230000000052 comparative effect Effects 0.000 abstract 1
Abstract
The present invention relates to a method for the comparative assessment of the level of specific nucleic acid sequences in samples derived from different sources. More specifically, the invention relates to a method using oligonucleotides covalently linked to a solid support, such as beads, to isolate specific labeled nucleic acid sequences from complex mixtures. The methods disclosed allow quantitative comparisons of the amount of nucleic acid of defined sequence in a plurality of different samples of nucleic acid, e.g., from different cells or tissues or from genetic libraries. Nucleic acids from the samples are labeled in such a fashion that the signals can be distinguished and compared following hybridization to the oligonucleotides on the beads. According to the invention, the solid supports with the hybridized nucleic acid may be retrieved, and the target nucleic acid eluted and analyzed. Furthermore, the invention provides a method for tagging individual clones from a cDNA library such that they can be identified uniquely and retrieved by hybridization to specific beads.
Claims
1. A method for comparing relative amounts of specific nucleic acid molecules in at least two samples, comprising the steps of:
(a) generating a target pool comprising a first and a second sample, wherein said first sample comprises nucleic acid molecules of a first source, and said first source nucleic acid molecules are linked to a first label, and wherein said second sample comprises nucleic acid molecules of a second source, and said second source nucleic acid molecules are linked to a second label;
(b) contacting said target pool with a plurality of solid supports each having attached thereto multiple capture oligonucleotides of a unique sequence under conditions which promote the formation of perfectly matched duplexes between said capture oligonucleotides and nucleic acid molecule complements within said target pool; and
(c) sorting the solid supports according to the relative amount of said first label and said second label; wherein the unique capture oligonucleotides attached to each solid support comprise a stretch of from about 10 to about 40 nucleotides of random sequence, or a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.
2. The method of Claim 1, further comprising the step of determining the identity of at least one nucleic acid molecule having said relative amount of said first and second labels of interest in step (c).
3. The method of Claim 1, wherein the nucleic acid molecules of said first and said second samples are derived from a first source and a second source, respectively, and wherein said first and second sources differ in cell type, tissue type, disease state or developmental stage.
4 The method of Claim 1, wherein the nucleic acid molecules in the target pool are derived from a first and a second source of genomic DNA. -101-
5. The method of Claim 1, wherein the nucleic acid molecules in the target pool are selected from the group consisting of mRNA and cDNA.
6. The method of Claim 5, wherein the nucleic acid molecules in the pool are cDNA molecules.
7. The method of Claim 6, wherein the cDNA molecules have attached thereto unique oligonucleotide identifier tags, each of said tags comprising a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.
8. The method of Claim 7, wherein the capture oligonucleotides attached to said solid supports comprise complements of said identifier tags.
9. The method of Claim 1, wherein the nucleic acid molecules of said first and said second sample are derived from cancerous and non-cancerous tissue, respectively.
10. The method of Claim 1, wherein the nucleic acid molecules of said first and said second sample are derived from plant cells, insect cells, fungal cells, bacterial cells, virus infected and uninfected cells, senescent and non-senescent cells, parental arrested cells and revertant growth proficient cells, or transgenic and normal cells.
11. The method of Claim 1, wherein the nucleic acid molecules of said first and said second sample are derived from cells before and after treatment with an agent, respectively.
12. The method of Claim 11, wherein the agent is selected from the group consisting of a naturally occurring growth factor, an immunologic factor, a therapeutic compound, a therapeutic lead compound, and a growth-arresting substance. -102-
13. The method of Claim 1, wherein the nucleic acid molecules of said first and said second sample are derived from a genetic library.
14. A method of normalizing a genetic library, comprising the steps of:
(a) attaching unique oligonucleotide identifier tags to cDNA inserts derived from a genetic library;
(b) hybridizing the cDNA inserts of step (a) with cDNA derived from a source of interest under conditions that promote the formation of perfectly matched duplexes, wherein the cDNA is labeled with a first label;
(c) contacting the mixture of step (b) with solid supports having attached thereto the complements of the oligonucleotide identifier tags under conditions that promote the formation of perfectly matched duplexes between the oligonucleotide identifier tags and their respective complements in the presence of free oligonucleotide identifier tags labeled with a second label and corresponding in sequence to the oligonucleotide identifier tags of step (a);
(d) sorting solid supports according to the relative amount of said first label and said second label, wherein said relative amount of said first and second label correlates with the abundance of a cDNA, or mRNA sequence; and
(e) amplifying cDNA inserts present at lower abundance in order to match the abundance of all cDNA sequences such that they are represented at substantially similar levels in the library.
15. The method of Claim 14, wherein the unique oligonucleotide identifier tag has a length of from about 14 to about 90 nucleotides.
16. The method of Claim 14, wherein the oligonucleotide identifier tag has a length of from about 16 to about 32 nucleotides.
17. The method of Claim 14, wherein the unique oligonucleotide identifier tag comprises a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides. -103-
18. A method for producing nucleic acid molecules having unique sequence identifier tags attached thereto comprising the steps of:
(a) generating sequence identifier tags comprising a combination of between -2 and 6 sequence units in tandem, each unit consisting of from about 7 to about 15 nucleotides; and
(b) attaching said sequence identifier tags to a population of nucleic acid molecules.
19. The method of Claim 18, further comprising the steps of either a) random priming or oligo(dT) priming with primers that comprise sequence identifier tags, or b) ligation of sequence identifier tag adapters onto said nucleic acid population; followed by cloning in E. coli or another host.
20. A method for comparing relative amounts of specific nucleic acid molecules in at least two samples wherein the relative abundance of one or more of said molecules changes during propagation in two host cell populations, comprising the steps of:
(a) introducing nucleic acid samples from a single source individually into non-identical cell populations;
(b) propagating the separate cell populations;
(c) re-isolating the nucleic acid samples from the separate cell populations; and
(d) subjecting the re-isolated nucleic acid samples to quantitative comparison of the relative amounts of at least one specific nucleic acid molecule.
21. A method for comparing relative amounts of specific nucleic acid molecules in at least two samples derived from a single nucleic acid source, wherein the relative abundance of one or more of said molecules changes during propagation in a host cell population, comprising the steps of:
(a) introducing a nucleic acid sample from a starting nucleic acid source into a cell population;
(b) propagating the cell population; -104-
(c) re-isolating the propagated nucleic acid sample from the cell population; and
(d) performing quantitative comparison of the relative amounts of at least one specific nucleic acid molecule in the propagated nucleic acid sample and the starting nucleic acid source.
22. The method of claim 20 or 21, further comprising an enrichment step (e), in which the samples from step (c) are subjected to one or more cycles of steps (a) through (c).
23. The method of claim 20 or 21 , wherein the single nucleic acid source is a genetic library.
24. The method of claim 23, wherein the library comprises inserts selected from the group consisting of genomic DNA, cDNA, and random sequence DNA.
25. The method of claim 23, wherein the genetic library comprises a plurality of inserts, the inserts comprising one or more sequences which, upon expression in a living host cell, are capable of differentially altering the phenotype of the host cell.
26. A method for identifying specific nucleic acid sequences in a genetic library whose relative abundance changes after propagation in two non-identical cell populations, the method comprising the steps of claim 20, and further comprising the step of:
(e) determining the identity of at least one nucleic acid molecule whose relative abundance differs after propagation in one cell population relative to another cell population.
27. A method for identifying specific nucleic acid sequences in a genetic library whose relative abundance increases or decreases during propagation in a host cell population, the method comprising the steps of claim 21, and further comprising the step of: -105-
(e) determining the identity of at least one nucleic acid molecule whose relative abundance differs after propagation in the cell population.
28. The method of Claim 2, 26 or 27, wherein the identity of the nucleic acid molecule is determined directly by DNA sequence analysis of the nucleic acids hybridized to the solid support.
29 The method of Claim 2, 26 or 27, wherein the identity of the nucleic acid molecule is determined indirectly by DNA sequence analysis of the oligonucleotide or fragment attached to the solid support.
30 The method of claim 20, 21, 26 or 27, wherein step (d) further comprises: (dl) differentially labeling nucleic acid samples derived from each library re- isolated from the cell populations to generate a first and a second labeled nucleic acid sample,
(d2) generating a target pool comprising said first and second nucleic acid samples,
(d3) contacting said target pool with a plurality of solid supports each having attached thereto multiple capture oligonucleotides of a unique sequence under conditions which promote the formation of perfectly matched duplexes between said capture oligonucleotides and nucleic acid molecule complements within said target pool, and
(d4) sorting the solid supports according to the relative amount of said first label and said second label.
31 The method of Claim 1 or 30, wherein the solid supports have attached thereto oligonucleotides complementary to nucleic acid molecules representing particular transcripts of interest.
32 The method of Claim 1 or 30, wherein the unique capture oligonucleotides attached to the solid supports have a length of from about 10 to about 50 nucleotides. -106-
33. The method of Claim 1 or 30, wherein the unique capture oligonucleotides attached to the solid supports have a length of from about 50 to about 5,000 nucleotides.
34. The method of Claim 1 or 30, wherein the unique capture oligonucleotides attached to the solid supports comprise a stretch of from about 10 to about 40 nucleotides of random sequence.
35. The method of Claim 1 or 30, wherein the unique capture oligonucleotides attached to the solid supports have a length of from about 12 to about 30 nucleotides and comprising a stretch of from about 10 to about 20 nucleotides of random sequence.
36. The method of Claim 1 or 30, wherein the unique capture oligonucleotides attached to the solid supports comprise a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.
37. The method of Claim 1 or 30, wherein the unique capture oligonucleotides attached to the solid supports comprise a stretch of from about 5 to about 25 adenosine residues at the 3' end, and a stretch of from about 8 to about 16 nucleotides of random sequence at the 5' end.
38. The method of Claim 1 or 30, wherein the target nucleic acid molecules have attached thereto unique oligonucleotide identifier tags, each of said tags comprising a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.
39. The method of Claim 38, wherein the capture oligonucleotides attached to said solid supports comprise complements of said unique identifier tags. -107-
40. The method of Claim 1, 14 or 41, wherein said first and said second label are distinguishable fluorescent labels.
41. The method of Claim 40, wherein said fluorescent labels are individually selected from the group consisting of 6-FAM, HEX, TET, TAMRA, ROX, JOE, 5- FAM, phycoerythrin and Rl 10.
42. The method of Claim 20, wherein the cell populations in step (a) are non- identical in phenotype.
43. The method of Claim 20, wherein the cell populations in step (a) are non- identical in genotype.
44. The method of Claim 20, wherein the cell populations in step (a) comprise cells which differ in cell type, tissue type, physiological state, disease state or developmental stage.
45. The method of Claim 20, wherein the cell populations in step (a) comprise cells before and after treatment with an agent, respectively.
46. The method of Claim 45, wherein the agent is selected from the group consisting of a naturally occurring growth factor, an immunologic factor, a small molecule compound of interest, a putative therapeutic compound, a therapeutic lead compound, and a growth-arresting substance.
47. The method of Claim 20, wherein the nucleic acid source is an expression library and the target nucleic acid molecules are RNA transcripts whose relative abundance represents the relative promoter activity of a specific nucleic acid sequence in the library after propagation in the non-identical cell populations.
48. The method of Claim 20, wherein the nucleic acid source comprises genomic DNA or random DNA sequence inserts and the relative abundance of the target nucleic -108-
acid molecules represents the relative differences in copy number of a specific nucleic " acid sequence in the library after propagation in the non-identical cell populations.
49 The method of Claim 21, wherein the nucleic acid source is an expression library and the target nucleic acid molecules are RNA transcripts whose relative abundance changes after propagation in the cell population.
50. The method of Claim 21, wherein the nucleic acid source comprises genomic or random sequence DNA inserts and the relative abundance of the target nucleic acid molecules represents the relative differences in copy number of a specific nucleic acid sequence in the library after propagation in the cell population.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU56057/98A AU5605798A (en) | 1996-12-13 | 1997-12-12 | Methods for measuring relative amounts of nucleic acids in complex mixture andretrieval of specific sequences therefrom |
| EP97952461A EP0948649A1 (en) | 1996-12-13 | 1997-12-12 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
| CA002275549A CA2275549A1 (en) | 1996-12-13 | 1997-12-12 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
| US10/053,366 US20030049599A1 (en) | 1996-12-13 | 2002-01-18 | Methods for negative selections under solid supports |
| US10/066,448 US20020172965A1 (en) | 1996-12-13 | 2002-02-01 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/764,191 | 1996-12-13 | ||
| US08/764,191 US6060240A (en) | 1996-12-13 | 1996-12-13 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
Related Parent Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US08/764,191 Continuation-In-Part US6060240A (en) | 1996-12-13 | 1996-12-13 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
| US08/764,191 Continuation US6060240A (en) | 1996-12-13 | 1996-12-13 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US32974799A Continuation-In-Part | 1996-12-13 | 1999-06-10 | |
| US33610799A Continuation-In-Part | 1996-12-13 | 1999-06-18 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1998026098A1 WO1998026098A1 (en) | 1998-06-18 |
| WO1998026098B1 true WO1998026098B1 (en) | 1998-08-27 |
Family
ID=25069951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/023173 WO1998026098A1 (en) | 1996-12-13 | 1997-12-12 | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US6060240A (en) |
| EP (1) | EP0948649A1 (en) |
| AU (1) | AU5605798A (en) |
| CA (1) | CA2275549A1 (en) |
| WO (1) | WO1998026098A1 (en) |
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