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US 12,509,719 B2Granted
Cell-free method of producing synthetic circular nucleic acid
AssigneeAldevron, LLC
InventorsNathan Russart, Venkata Indurthi, Denis Arutyunov
Life of this patent
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Abstract
The present disclosure relates to a process of cell-free circular nucleic acid amplification using rolling circle amplification.
Claims13
A method of amplifying a synthetic circular nucleic acid, comprising:
- a) providing a circular nucleic acid template comprising:
- (i) a backbone, wherein the backbone comprises one or more internal endonuclease cut sites, and
- (ii) an insert sequence, wherein the insert sequence comprises endonuclease cut sites on its 5′ and 3′ terminal ends;
- b) amplifying the circular nucleic acid template by rolling circle amplification, thereby producing an amplification product;
- c) contacting the amplification product with a first endonuclease that cuts at the endonuclease cut sites at the 5′ and 3′ ends of the insert sequence under conditions for digestion, thereby producing a first digestion product;
- d) adding the first digestion product to a ligation reaction mixture comprising a ligase enzyme, wherein the first digestion product is added to the ligation reaction mixture at a rate of about 1% to about 20% of a final ligation reaction volume per hour over a 5 to 14 hour period;
- e) incubating the ligation reaction mixture, thereby producing a circular ligation product, wherein the circular ligation product is at least partially supercoiled;
- f) contacting the circular ligation product with an exonuclease that digests single-stranded nucleic acid, linear nucleic acid, and open circle nucleic acid, thereby producing a final reaction product comprising synthetic circular nucleic acid that is substantially free of linear nucleic acid;
- and
- g) purifying the final reaction product by ion-exchange chromatography, affinity chromatography, reverse phase chromatography, size exclusion chromatography, isopropanol precipitation, methanol precipitation, ethanol precipitation, solid phase purification, electrophoresis, or combinations thereof.
7 more elements
A method of amplifying a synthetic circular nucleic acid, comprising:
- a) providing a circular nucleic acid template comprising
- (i) a backbone, wherein the backbone comprises one or more internal endonuclease cut sites, and
- (ii) an insert sequence, wherein the insert sequence comprises endonuclease cut sites on its 5′ and 3′ terminal ends;
- b) amplifying the circular nucleic acid template by rolling circle amplification, wherein the rolling circle amplification is performed using a Phi29 DNA polymerase, thereby producing an amplification product;
- c) contacting the amplification product with a first endonuclease targeting the endonuclease cut sites on the insert sequence, under conditions for digestion, wherein the first endonuclease is a Type II restriction endonuclease, thereby producing a first digestion product;
- d) adding the first digestion product to a ligation reaction mixture comprising a T4 DNA ligase wherein the first digestion product is added to the ligation reaction mixture at a rate of about 1% to about 20% of a final ligation reaction volume per hour over a 5-14-hour period;
- e) incubating the ligation reaction mixture, thereby producing a circular ligation product;
- f) contacting the circular ligation product with a second endonuclease targeting the one or more internal endonuclease cut sites on the backbone, under conditions for digestion, wherein the second endonuclease is a Type II restriction endonuclease, thereby producing a second digestion product, wherein the first and second endonucleases do not have the same recognition site;
- g) contacting the second digestion product with a T5 exonuclease that digests single-stranded nucleic acid, linear nucleic acid, and open circle nucleic acid, thereby producing a final reaction product comprising synthetic circular nucleic acid that is substantially free of linear nucleic acid;
- and
- h) purifying the final reaction product by ion-exchange chromatography, wherein the final reaction product is at least partially supercoiled.
8 more elements
Dependencies are inferred from the claim text.