Rolling circle replication reporter systems

• US 6,344,329 B1
• Filed: 08/23/2000
• Issued: 02/05/2002
• 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:
  US09/644,723
• Time in Patent Office: 531 Days
• Field of Search: 436/6, 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 method of selectively amplifying nucleic acid sequences related to one or more target sequences, the method comprising,

(a) mixing one or more different open circle probes with a target sample, 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 primer 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, 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 replication of the amplification target circles,

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

wherein at least one of the target sequences is part of a reporter binding agent.

2. The method of claim 1 wherein the reporter binding agent comprises an amplification target circle.

3. The method of claim 1 further comprising detecting the presence of tandem sequence DNA, or measuring the amount of tandem sequence DNA formed.

4. A method of amplifying nucleic acid sequences, the 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 replication of the circularized open circle probe and secondary DNA strand displacement.

5. The method of claim 4 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

6. A method for detecting an analyte in a sample, the method comprising

an amplification operation,

wherein a nucleic acid tag is coupled to a specific binding molecule,

wherein the amplification operation comprises amplification of the nucleic acid tag via rolling circle replication of an amplification target circle to produce tandem sequence DNA, and

detecting the tandem sequence DNA.

7. The method of claim 6 wherein the specific binding molecule interacts with a target molecule.

8. The method of claim 6 wherein the specific binding molecule is bound to a target molecule.

9. The method of claim 6 wherein the specific,binding molecule is an antibody.

10. The method of claim 6 wherein the nucleic acid tag is an amplification target circle, wherein the amplification target circle is tethered to the specific binding molecule.

11. A method for in situ detection of a target nucleic acid in a sample comprising the steps of:

(a) preparing a target sample to be analyzed for the presence of a target nucleic acid;

(b) washing the target sample;

(c) adding an open circle probe resulting in hybridization between the open circle probe and the target nucleic acid;

(d) ligating the open circle probes to form an amplification target circle;

(e) washing the amplification target circle and target nucleic acid;

(f) amplifying the amplification target circle by

mixing the amplification target circle with a rolling circle replication primer, wherein the amplification target circle comprises a spacer region comprising a primer complement portion, wherein the rolling circle replication primer is complementary and hybridizes to the primer complement portion, a secondary DNA strand displacement primer, wherein the secondary DNA strand displacement primer does not match the primer complement portion, a DNA polymerase capable of strand displacement and dNTPs, and

incubating under conditions that promote replication of the amplification target circle to form tandem sequence DNA and that promote hybridization between the tandem sequence DNA and secondary DNA strand displacement primer and replication of the tandem sequence DNA to form secondary tandem sequence DNA,

whereby replication of the tandem sequence DNA displaces other strands being synthesized downstream and the rolling circle replication primer can hybridize to and primer replication of the secondary tandem sequence DNA to form tertiary tandem sequence DNA;

(g) allowing the amplification to proceed until multiple copies of secondary tandem sequence DNA and tertiary tandem sequence DNA are formed;

(h) detecting the secondary tandem sequence DNA and tertiary tandem sequence DNA, wherein detection of the secondary tandem sequence DNA and tertiary tandem sequence DNA indicates presence of the target nucleic acid in the target sample.

12. A method for in situ detection of a target nucleic acid in a sample comprising the steps of:

(a) preparing a target sample to be analyzed for the presence of a target nucleic acid;

(b) washing the target sample;

(c) adding an open circle probe resulting in hybridization between the open circle probe and the target nucleic acid;

(d) ligating the open circle probes to form an amplification target circle;

(e) washing the amplification target circle and target nucleic acid;

(f) amplifying the amplification target circle by

mixing the amplification target circle with a rolling circle replication primer, wherein the amplification target circle comprises a spacer region comprising a primer complement portion, wherein the rolling circle replication primer is complementary and hybridizes to the primer complement portion, a DNA polymerase capable of strand displacement, and dNTPs, and

incubating under conditions that promote replication of the amplification target circle to form tandem sequence DNA;

(g) detecting the tandem sequence DNA, wherein detection of the tandem sequence DNA indicates presence of the target nucleic acid in the target sample.