WO2005094532B1 - Encoding and decoding reactions for determining target polynucleotides - Google Patents
Encoding and decoding reactions for determining target polynucleotidesInfo
- Publication number
- WO2005094532B1 WO2005094532B1 PCT/US2005/009882 US2005009882W WO2005094532B1 WO 2005094532 B1 WO2005094532 B1 WO 2005094532B1 US 2005009882 W US2005009882 W US 2005009882W WO 2005094532 B1 WO2005094532 B1 WO 2005094532B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- primer
- encoding
- reverse
- target polynucleotide
- labeling probe
- Prior art date
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract 39
- 229920000023 polynucleotide Polymers 0.000 title claims abstract 30
- 239000002157 polynucleotide Substances 0.000 title claims abstract 30
- 230000003321 amplification Effects 0.000 claims abstract 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract 9
- 239000007795 chemical reaction product Substances 0.000 claims abstract 5
- 239000000523 sample Substances 0.000 claims 37
- 238000002372 labelling Methods 0.000 claims 30
- 239000000047 product Substances 0.000 claims 18
- 230000000295 complement Effects 0.000 claims 16
- 239000000203 mixture Substances 0.000 claims 8
- 101700080605 NUC1 Proteins 0.000 claims 3
- 101700006494 nucA Proteins 0.000 claims 3
- 108020004999 Messenger RNA Proteins 0.000 claims 2
- 229920002106 messenger RNA Polymers 0.000 claims 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 230000001809 detectable Effects 0.000 abstract 1
Abstract
The present invention is directed to methods, reagents, and kits for detecting the presence or absence of (or quantifying) target polynucleotide sequences in at least one sample using encoding and decoding reactions. When a particular target polynucleotide is present in a sample, a reaction product is formed in the encoding reaction that includes addressable primer portions. At least one label and at least one address primer is employed in the decoding amplification reaction thereby providing a detectable signal value depending upon whether a sequence is present or absent. In some embodiments universal bases and reduced libraries of probes can be employed for highly multiplexed analysis of target polynucleotides.
Claims
1. (canceled)
2. (new) A method for comparing the amount of a target polynucleotide sequence between two samples comprising; providing a first reaction vessel one and a first reaction vessel two; wherein the first reaction vessel one comprises a first sample, a forward primer, and a reverse primer, wherein the forward primer comprises a target specific portion and a forward addressable primer portion, wherein the reverse primer comprises a target specific portion and a reverse addressable primer portion, wherein . the forward primer or the reverse primer further comprises a first identifying portion; wherein the first reaction vessel two comprises a second sample, a forward primer and a reverse primer, wherein the forward primer comprises a target specific portion and a forward addressable primer portion, wherein the reverse primer comprises a target specific portion and a reverse addressable primer portion, wherein the forward primer or the reverse primer further comprises a seqond identifying portion; performing a first encoding PCR in the first reaction vessel one, thereby forming a first encoding PCR product mixture comprising a first encoding PCR product, wherein the first encoding PCR product comprises the forward addressable primer portion, the first identifying portion, the target specific portions, and the reverse addressable primer portion, wherein the identity of target polynucleotide sequence is encoded with the forward addressable primer portion and the reverse addressable primer portion, and wherein the identity of the sample in the first encoding PCR is encoded by the first identifying portion; performing a second encoding PCR in the first reaction vessel two, thereby forming a second encoding PCR product mixture comprising a second encoding PCR product, wherein the second encoding PCR product comprises the forward addressable primer portion, the second identifying portion, the target specific portions, and the reverse addressable primer portion, wherein the identity of the target polynucleotide sequence is encoded with the forward addressable primer portion and the reverse addressable primer portion, and wherein the identity of the sample in the second encoding PCR is encoded by the second identifying portion;
84 combining the first encoding PCR product mixture from the first reaction vessel one with the second encoding PCR product mixture from the first reaction vessel two to form a combined encoding PCR product mixture; providing a second reaction vessel, wherein the second reaction vessel comprises a forward address primer, a reverse address primer, a first labeling probe, and a second labeling probe, wherein the first labeling probe one is complementary to, or complementary to the complement of, the first identifying portion of the encoding PCR product from the first reaction vessel one, and wherein the second labeling probe one is complementary to, or complementary to the complement of, the second identifying portion of the encoding PCR product from the first reaction vessel two; adding an aliquot of the combined encoding PCR product mixture to the second reaction vessel, thereby forming a decoding amplification reaction in the second reaction vessel; performing a decoding amplification reaction in the second reaction vessel, wherein the forward address primer hybridizes to the complement of the forward addressable primer portion introduced into the encoding PCR product during the first encoding PCR and the second encoding PCR, wherein the reverse address primer hybridizes to the reverse addressable primer portion introduced in the encoding PCR product during the first encoding PCR and the second encoding PCR, wherein amplification of the encoding PCR product results in signal from the first labeling probe when the encoding PCR product is derived from the first encoding PCR, wherein amplification of the encoding PCR product results in signal from the second labeling probe when the encoding PCR product is derived from the second encoding PCR; detecting and quantifying the target polynucleotide in the two samples based on the presence and quantity of signal from the first labeling probe and the second labeling probe in the decoding reaction; and, comparing the amount of the target polynucleotide sequence between two samples.
3. (new) The method according to claim 2 wherein a plurality of target polynucleotide sequences are compared between two samples; said method further comprising; a plurality of different forward primers and reverse primers in the first encoding PCR that are specific for the plurality of target polynucleotide sequences;
85 a plurality of different forward primers and reverse primers in the second encoding PCR that are specific for the plurality of target polynucleotide sequences; wherein the plurality of different forward and reverse primers in the first encoding PCR comprise a distinct forward addressable primer portion, a distinct reverse addressable primer portion, and the same first identifying portion; wherein the plurality of different forward and reverse primers in the second encoding PCR comprise a distinct forward addressable primer portion, a distinct reverse addressable primer portion, and the same second identifying portion; wherein the distinct forward addressable primer portion and distinct reverse addressable primer portion are the same for a given target polynucleotide between the two samples; performing a plurality of decoding amplification reactions in a plurality of second reaction vessels; detecting and quantifying the plurality of target polynucleotide in the two samples based on the presence and quantity of signal from the first labeling probe and the second labeling probe in each decoding reaction; and, comparing the amount of a plurality of target polynucleotide sequences between two samples.
4. (new) The method according to claim 2 wherein the first labeling probe, the second labeling probe, or both, comprise PNA.
5. (new) The method according to claim 2 wherein the first labeling probe, the second labeling probe, or both, comprise a 5' nuclease cleavable probe.
6. (new) The method according to claim 2 wherein the target polynucleotide sequences are messenger RNA.
7. (new) A method for comparing the amount of a target polynucleotide sequence between two samples comprising; performing a first encoding reaction on a target polynucleotide sequence to form a first encoding reaction product, wherein the identity of the target polynucleotide sequence is encoded with addressable primer portions and wherein the identity of the sample is encoded with a first identifying portion;
86 performing a second encoding reaction on a target polynucleotide to form a second encoding reaction product, wherein the identity of the target polynucleotide sequence is encoded with addressable primer portions and wherein the identity of the sample is encoded with a second identifying portion; combining the first encoding reaction product with the second encoding reaction product; performing a decoding amplification reaction, wherein the decoding amplification reaction comprises a PCR comprising a first labeling probe and a second labeling probe, wherein first labeling probe is complementary to, or complementary to the complement of, the first identifying portion, and wherein second labeling probe is complementary to, or complementary to the complement of, the second identifying portion; and, comparing the amount of the target polynucleotide sequence between the two samples.
8. (new) The method according to claim 7 wherein the first encoding reaction is a PCR and wherein the second encoding reaction is a PCR.
9. (new) The method according to claim 8 wherein a plurality of target polynucleotide sequences are compared between two samples; said method further comprising; a plurality of different forward primers and reverse primers in the first encoding PCR that are specific for the plurality of target polynucleotide sequences; a plurality of different forward primers and reverse primers in the second encoding PCR that are specific for the plurality of target polynucleotide sequences; wherein the plurality of different forward and reverse primers in the first encoding PCR comprise a distinct forward addressable primer portion, a distinct reverse addressable primer portion, and the same first identifying portion; wherein the plurality of different forward and reverse primers in the second encoding PCR comprise a distinct forward addressable primer portion, a distinct reverse addressable primer portion, and the same second identifying portion; wherein the distinct forward addressable primer portion and distinct reverse addressable primer portion are the same for a given target polynucleotide between the two samples;
87 performing a plurality of decoding amplification reactions in a plurality of second reaction vessels; detecting and quantifying the plurality of target polynucleotide in the two samples based on the presence and quantity of signal from the first labeling probe and the second labeling probe in each decoding reaction; and, comparing the amount of a plurality of target polynucleotide sequences between two samples.
10. (new) The method according to claim 7 wherein the first encoding reaction is a ligation reaction and wherein the second encoding reaction is a ligation reaction.
11. (new) The method according to claim 7 wherein the first labeling probe, the second labeling probe, or both, comprise PNA.
12. (new) The method according to claim 7 wherein the first labeling probe, the second labeling probe, or both, are 51 nuclease cleavable.
13. (new) The method according to claim 7 wherein the target polynucleotide sequences are messenger RNA.
14. (new) A kit for comparing the amount of a target polynucleotide sequence between two samples comprising; a forward primer, wherein the forward primer comprises a target specific portion and a forward addressable primer portion; a reverse primer, wherein the reverse primer comprises a target specific portion and a reverse addressable primer portion; wherein the forward primer, the reverse primer, or both, further comprise an identifying portion; a reaction vessel comprising a forward address primer, a reverse address primer, and a labeling probe, wherein the labeling probe is complementary to, or complementary to the complement of, the identifying portion; and, a PCR master mix, wherein the PCR master mix comprises a polymerase, dNTPs, and a buffer.
88
15. (new) The kit according to claim 14 wherein the first labeling probe, the second labeling probe, or both, comprise PNA.
16. (new) The kit according to claim 14 wherein the first labeling probe, the second labeling probe, or both, are 5' nuclease cleavable.
89
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007505187A JP2007530048A (en) | 2004-03-24 | 2005-03-24 | Encoding and decoding reactions to detect target polynucleotides |
| DE602005007874T DE602005007874D1 (en) | 2004-03-24 | 2005-03-24 | CODING AND DECODING REACTIONS FOR DETERMINING TARGET POLYNUCLEOTIDES |
| EP05732398A EP1730312B1 (en) | 2004-03-24 | 2005-03-24 | Encoding and decoding reactions for determining target polynucleotides |
Applications Claiming Priority (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US55622404P | 2004-03-24 | 2004-03-24 | |
| US55616204P | 2004-03-24 | 2004-03-24 | |
| US55616304P | 2004-03-24 | 2004-03-24 | |
| US55615704P | 2004-03-24 | 2004-03-24 | |
| US60/556,224 | 2004-03-24 | ||
| US60/556,163 | 2004-03-24 | ||
| US60/556,157 | 2004-03-24 | ||
| US60/556,162 | 2004-03-24 | ||
| US63068104P | 2004-11-24 | 2004-11-24 | |
| US60/630,681 | 2004-11-24 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO2005094532A2 WO2005094532A2 (en) | 2005-10-13 |
| WO2005094532A3 WO2005094532A3 (en) | 2005-12-22 |
| WO2005094532B1 true WO2005094532B1 (en) | 2006-03-02 |
Family
ID=35064420
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2005/010184 WO2005098040A2 (en) | 2004-03-24 | 2005-03-24 | Ligation and amplification reactions for determining target molecules |
| PCT/US2005/009882 WO2005094532A2 (en) | 2004-03-24 | 2005-03-24 | Encoding and decoding reactions for determining target polynucleotides |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2005/010184 WO2005098040A2 (en) | 2004-03-24 | 2005-03-24 | Ligation and amplification reactions for determining target molecules |
Country Status (6)
| Country | Link |
|---|---|
| US (2) | US7601821B2 (en) |
| EP (3) | EP1730312B1 (en) |
| JP (2) | JP2007530048A (en) |
| AT (2) | ATE431433T1 (en) |
| DE (2) | DE602005007874D1 (en) |
| WO (2) | WO2005098040A2 (en) |
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2005
- 2005-03-24 WO PCT/US2005/010184 patent/WO2005098040A2/en not_active Application Discontinuation
- 2005-03-24 JP JP2007505187A patent/JP2007530048A/en active Pending
- 2005-03-24 DE DE602005007874T patent/DE602005007874D1/en active Active
- 2005-03-24 WO PCT/US2005/009882 patent/WO2005094532A2/en active Search and Examination
- 2005-03-24 US US11/090,830 patent/US7601821B2/en not_active Expired - Fee Related
- 2005-03-24 AT AT05760637T patent/ATE431433T1/en not_active IP Right Cessation
- 2005-03-24 EP EP05732398A patent/EP1730312B1/en not_active Not-in-force
- 2005-03-24 JP JP2007505241A patent/JP2007530051A/en active Pending
- 2005-03-24 US US11/090,468 patent/US7604937B2/en not_active Expired - Fee Related
- 2005-03-24 AT AT05732398T patent/ATE399884T1/en not_active IP Right Cessation
- 2005-03-24 DE DE602005014453T patent/DE602005014453D1/en active Active
- 2005-03-24 EP EP09004414.0A patent/EP2161346B1/en not_active Not-in-force
- 2005-03-24 EP EP05760637A patent/EP1727913B8/en not_active Not-in-force
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