Rolling circle replication reporter systems

• US 6,183,960 B1
• Filed: 08/11/1998
• Issued: 02/06/2001
• Est. Priority Date: 11/21/1995
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Lizardi, Paul M.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/132,552
• Time in Patent Office: 910 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: C12Q 2545/10 the purpose being quantitative analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting 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 a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, 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 the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

Claims

1. A kit for selectively amplifying nucleic acid sequences related to one or more target sequences, each comprising a 5′

region and a 3′

region, the kit comprising,

(a) one or more open circle probes each comprising a single-stranded, linear DNA molecule comprising, from 5′

end to 3′

end, a 5′

phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′

hydroxyl group,

wherein the spacer portion comprises a primer complement portion, and

wherein the left target probe portion is complementary to the 3′

region of at least one of the target sequences and the right target probe portion is complementary to the 5′

region of the same target sequence,

(b) a rolling circle replication primer comprising a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the open circle probes, and

(c) a secondary DNA strand displacement primer comprising a single-stranded, linear nucleic acid molecule comprising a matching portion that matches a portion of one or more of the open circle probes.

2. A kit for selectively detecting a target molecule, the kit comprising,

(a) one or more amplification target circles,

wherein the amplification target circle comprises a single-stranded, circular DNA molecule comprising a primer complement portion, wherein each amplification target circle is tethered to a specific binding molecule so that the amplification target circle can rotate freely, and

(b) a rolling circle replication primer comprising a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the amplification target circles.

3. The kit of claim 2 further comprising a secondary DNA strand displacement primer comprising a single-stranded, linear nucleic acid molecule comprising a matching portion that matches a portion of one or more of the amplification target circles.

4. The kit of claim 2 wherein the amplification target circle is tethered to a specific binding molecule via a tether loop.

5. A kit for selectively amplifying nucleic acid sequences related to one or more target sequences, each comprising a 5′

region and a 3′

region, the kit comprising,

(a) one or more open circle probes each comprising a single-stranded, linear DNA molecule comprising, from 5′

end to 3′

end, a 5′

phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′

hydroxyl group,

wherein the spacer portion comprises a primer complement portion, and

wherein the left target probe portion is complementary to the 3′

region of at least one of the target sequences and the right target probe portion is complementary to the 5′

region of the same target sequence,

(b) a rolling circle replication primer comprising a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the open circle probes, and

(c) one or more reporter agents each comprising a specific binding molecule and an oligonucleotide portion, wherein the oligonucleotide portion comprises one of the target sequences.

6. A kit for selectively amplifying nucleic acid sequences related to one or more target sequences, each comprising a 5′

region and a 3′

region, the kit comprising,

(a) one or more open circle probes each comprising a single-stranded, linear DNA molecule comprising, from 5′

end to 3′

end, a 5′

phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′

hydroxyl group,

wherein the spacer portion comprises a primer complement portion, and

wherein the left target probe portion is complementary to the 3′

region of at least one of the target sequences and the right target probe portion is complementary to the 5′

region of the same target sequence,

wherein the target sequence further comprises a central region located between the 5′

region and the 3′

region,

wherein neither the left target probe portion of the open circle probe nor the right target probe portion of the open circle probe is complementary to a central region of the target sequence,

(b) a rolling circle replication primer comprising a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the open circle probes,

(c) one or more gap oligonucleotides, wherein the gap oligonucleotides are complementary to all or a portion of the central region of the target sequence, and

(d) a secondary DNA strand displacement primer comprising a single-stranded, linear nucleic acid molecule comprising a matching portion that matches a portion of one or more of the open circle probes.

7. An amplification target circle, wherein the amplification target circle comprises a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the amplification target circle is tethered to a specific binding molecule via a tether loop so that the amplification target circle can rotate freely.