CA2236161A1 - Unimolecular segment amplification and detection - Google Patents
Unimolecular segment amplification and detectionInfo
- Publication number
- CA2236161A1 CA2236161A1 CA 2236161 CA2236161A CA2236161A1 CA 2236161 A1 CA2236161 A1 CA 2236161A1 CA 2236161 CA2236161 CA 2236161 CA 2236161 A CA2236161 A CA 2236161A CA 2236161 A1 CA2236161 A1 CA 2236161A1
- Authority
- CA
- Canada
- Prior art keywords
- amplification
- target
- molecules
- sample
- rolling circle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000003321 amplification Effects 0.000 title abstract 9
- 238000003199 nucleic acid amplification method Methods 0.000 title abstract 9
- 238000001514 detection method Methods 0.000 title abstract 4
- 239000000523 sample Substances 0.000 abstract 9
- 238000000034 method Methods 0.000 abstract 5
- 230000010076 replication Effects 0.000 abstract 4
- 238000005096 rolling process Methods 0.000 abstract 4
- 150000007523 nucleic acids Chemical class 0.000 abstract 3
- 102000039446 nucleic acids Human genes 0.000 abstract 3
- 108020004707 nucleic acids Proteins 0.000 abstract 3
- 230000004543 DNA replication Effects 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 239000000203 mixture Substances 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal.
A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one ore more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe molecules to a target molecule present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. A single round of amplification using rolling circle replication results in a large amplification of the amplification target circles. Following rolling circle replication, the amplified sequences are detected using combinatorial multicolor coding probes that allow separate, simultaneous, and quantitative detection of multiple different amplified target circles representing multiple different target molecules.
Since the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. It is also advantageous that the DNA replication step is isothermal, and that signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme.
A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one ore more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe molecules to a target molecule present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. A single round of amplification using rolling circle replication results in a large amplification of the amplification target circles. Following rolling circle replication, the amplified sequences are detected using combinatorial multicolor coding probes that allow separate, simultaneous, and quantitative detection of multiple different amplified target circles representing multiple different target molecules.
Since the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. It is also advantageous that the DNA replication step is isothermal, and that signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US563,912 | 1983-12-21 | ||
| US08/563,912 US5854033A (en) | 1995-11-21 | 1995-11-21 | Rolling circle replication reporter systems |
| US1667796P | 1996-05-01 | 1996-05-01 | |
| US60/016,677 | 1996-05-01 | ||
| PCT/US1996/018812 WO1997019193A2 (en) | 1995-11-21 | 1996-11-21 | Unimolecular segment amplification and detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2236161A1 true CA2236161A1 (en) | 1997-05-29 |
| CA2236161C CA2236161C (en) | 2007-10-30 |
Family
ID=29407576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2236161 Expired - Lifetime CA2236161C (en) | 1995-11-21 | 1996-11-21 | Unimolecular segment amplification and detection |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2236161C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9683255B2 (en) | 2005-09-09 | 2017-06-20 | Qiagen Gmbh | Method for activating a nucleic acid for a polymerase reaction |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9487823B2 (en) | 2002-12-20 | 2016-11-08 | Qiagen Gmbh | Nucleic acid amplification |
| US7955795B2 (en) | 2003-06-06 | 2011-06-07 | Qiagen Gmbh | Method of whole genome amplification with reduced artifact production |
-
1996
- 1996-11-21 CA CA 2236161 patent/CA2236161C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9683255B2 (en) | 2005-09-09 | 2017-06-20 | Qiagen Gmbh | Method for activating a nucleic acid for a polymerase reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2236161C (en) | 2007-10-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20161121 |