Rolling circle amplification of RNA
First Claim
1. A method of amplifying RNA sequences, the method comprising incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, incubating a circularization probe and the first strand cDNA molecules under conditions that promote circularization of the first strand cDNA molecules, and incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication of the circularized first strand cDNA molecules, thereby amplifying RNA sequences.
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Abstract
Disclosed are compositions and methods for amplification of RNA molecules. The disclosed method involves synthesizing first strand cDNA molecules from RNA molecules, circularizing the first strand cDNA molecules and replicating the circularized first strand cDNA molecules using rolling circle replication. The method can be aided by the use of specialized primers for cDNA synthesis and specialized probes for circularizing the first strand cDNA molecules. The method can be used to replicate and amplify multiple RNA molecules, such as all RNA molecules in a sample or all mRNA molecules in a sample, or be used to replicate and amplify specific RNA molecules. Rolling circle replication of the circularized first strand cDNA molecules results in long DNA strands containing tandem repeats of the cDNA sequence. The tandem sequence DNA can be used directly (for detection of sequences, for example), further amplified, or used any other purpose. Double-stranded tandem sequence DNA can be used to produce unit lengths of the cDNA sequence. Tandem sequence DNA can also be transcribed to produce transcripts having sequence complementary to or matching the sequence of RNA molecules.
138 Citations
179 Claims
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1. A method of amplifying RNA sequences, the method comprising
incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, incubating a circularization probe and the first strand cDNA molecules under conditions that promote circularization of the first strand cDNA molecules, and incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication of the circularized first strand cDNA molecules, thereby amplifying RNA sequences.
- 68. The method of claim 68 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
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174. A method of amplifying RNA sequences, the method comprising
incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, wherein the conditions that promote synthesis of first strand cDNA molecules comprise incubating the cDNA primer and the RNA sample in the presence of a reverse transcriptase, incubating the first strand cDNA molecules in the presence of an RNAse H activity, incubating the first strand cDNA molecules under alkaline conditions, neutralizing the first strand cDNA molecules, purifying the first strand cDNA molecules, incubating a circularization probe and the first strand cDNA molecules under conditions that promote circularization of the first strand cDNA molecules, wherein the conditions that promote circularization of the first strand cDNA molecules comprise incubating the circularization probe and the first strand cDNA molecules in the presence of ligase, incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication of the circularized first strand cDNA molecules, thereby amplifying RNA sequences, wherein the conditions that promote rolling circle replication of the circularized first cDNA molecules comprise incubating the circularized first strand cDNA molecules in the presence of a DNA polymerase.
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176. A method of amplifying RNA sequences, the method comprising
incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, incubating a circularization probe and the first strand cDNA molecules under conditions that promote ligation of the first strand cDNA molecules to each other to form first strand cDNA concatemers, and incubating the first strand cDNA concatemers under conditions that promote strand displacement replication of the first strand cDNA concatemers, thereby amplifying RNA sequences.
Specification
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- Resources
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Current AssigneeQiagen GmbH (Qiagen NV)
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Original AssigneeQiagen GmbH (Qiagen NV)
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InventorsKumar, Gyanendra, Abarzua, Patricio
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current435/6
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CPC Class CodesC12N 15/1096 cDNA Synthesis; Subtracted ...C12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6844 Nucleic acid amplification ...C12Q 2525/131 incorporating a restriction...C12Q 2531/125 Rolling circle
