Rolling circle replication reporter systems

• US 6,797,474 B2
• Filed: 01/02/2002
• Issued: 09/28/2004
• Est. Priority Date: 11/21/1995
• Status: Expired due to Fees

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Lizardi, Paul M.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US10/038,718
• Publication Number:
  US 20030032024A1
• Time in Patent Office: 1,000 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.5
• 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 method comprising

a DNA ligation operation and an amplification operation,

wherein the DNA ligation operation comprises circularization of an open circle probe, wherein circularization of the open circle probe is dependent on hybridization of the open circle probe to a target sequence,

wherein the amplification operation comprises rolling circle amplification of the circularized open circle probe to produce tandem sequence DNA, wherein rolling circle amplification is primed by a rolling circle replication primer, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer,

wherein the rolling circle replication primer and tertiary DNA strand displacement primer are complementary to the circularized open circle probe, wherein the secondary DNA strand displacement primer matches sequence on the circularized open circle probe, wherein the secondary DNA strand displacement primer is not complementary to the complement of either the rolling circle replication primer or the tertiary DNA strand displacement primer.

2. The method of claim 1 wherein the target sequence is coupled to a specific binding molecule, wherein the specific binding molecule can interact with a target molecule.

3. The method of claim 2 wherein the target molecule is a protein.

4. The method of claim 2 wherein the target molecule is a nucleic acid molecule.

5. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more open circle probes with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture,

(b) mixing ligase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles,

(c) mixing a rolling circle replication primers, a secondary DNA strand displacement primers, and a tertiary DNA strand displacement primers with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture,

wherein the rolling circle replication primer and tertiary DNA strand displacement primer are complementary to one or more of the amplification target circles, wherein the secondary DNA strand displacement primers match sequence on one or more of the amplification target circles, wherein the secondary DNA strand displacement primers are not complementary to the complement of the rolling circle replication primers or the tertiary DNA strand displacement primers, and

(d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote amplification of the amplification target circles,

wherein amplification of the amplification target circle results in the formation of tandem sequence DNA.

6. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing a rolling circle replication primer, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule, wherein the rolling circle replication primers and tertiary DNA strand displacement primers are complementary to one or more of the amplification target circles, wherein the secondary DNA strand displacement primers match sequence on one or more of the amplification target circles, wherein the secondary DNA strand displacement primers are not complementary to the complement of the rolling circle replication primers or the tertiary DNA strand displacement primers, and

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote amplification of the amplification target circles,

wherein amplification of the amplification target circles results in the formation of tandem sequence DNA.

7. A method for detecting target molecules, the method comprising

an amplification operation,

wherein a reporter binding agent and a target molecule are brought into contact, wherein the reporter binding agent comprises a specific binding molecule and an oligonucleotide, wherein the specific binding molecule interacts with the target molecule, wherein an amplification target circle and the reporter binding agent are brought into contact,

wherein the amplification operation comprises rolling circle amplification of the amplification target circle to produce tandem sequence DNA, wherein rolling circle amplification is primed by the oligonucleotide of the reporter binding agent, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer and detecting the presence of tandem sequence DNA, thereby detecting the target molecule.

8. A method for detecting target molecules, the method comprising

an amplification operation,

wherein a reporter binding agent and a target molecule are brought into contact, wherein the reporter binding agent comprises a specific binding molecule and an oligonucleotide, wherein the oligonucleotide comprises an amplification target circle, wherein the specific binding molecule interacts with the target molecule,

wherein the amplification operation comprises rolling circle amplification of the amplification target circle to produce tandem sequence DNA, wherein rolling circle amplification is primed by a rolling circle replication primer, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer,

wherein the rolling circle replication primer and tertiary DNA strand displacement primer are complementary to the amplification target circle, wherein the rolling circle replication primer and tertiary DNA strand displacement primer are not complementary to the same sequence on the amplification target circle, wherein the secondary DNA strand displacement primer matches sequence on the amplification target circle, wherein the secondary DNA strand displacement primer is not complementary to the complement of either the rolling circle replication primer or the tertiary DNA strand displacement primer and detecting the presence of tandem sequence DNA, thereby detecting the target molecule.