Amplification of nucleic acids
AssigneeCofactor Genomics, Inc.
InventorsJon R. Armstrong, Jeffrey F. Hiken
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Abstract
Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.
Claims45
A method of amplifying a nucleic acid, the method comprising:
- i. priming a circular RNA molecule with one or more DNA primers;
- and
- ii. extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule.
A method of constructing a circular cDNA molecule, the method comprising ligating with a ligase one or more linear cDNA fragments bound to a circular RNA molecule scaffold, wherein the one or more linear cDNA fragments and the circular RNA molecule scaffold form an RNA-DNA heteroduplex, to convert the one or more linear cDNA fragments into a covalently closed circular cDNA molecule, thereby constructing a circular cDNA molecule.
A kit for use in a method of constructing a circular DNA molecule, the kit comprising a ligase with the ability to ligate adjacent 5′ and 3′ DNA ends that are bound to RNA in an RNA-DNA heteroduplex, and instructions for use of the kit.
Dependencies are inferred from the claim text.