Cell Free Biosynthesis of High-Quality Nucleic Acid and Uses Thereof
First Claim
1. A process for producing high-quality nucleic acid in a cell free system, comprising:
- (a) combining a circular template in a reaction mixture with one or more primers, which are complementary to at least one strand of the circular template, to form a template-primer complex;
(b) incubating the template-primer complex with at least one high-fidelity nucleic acid polymerase to produce a concatamer comprising tandem units of the circular template; and
(c) cutting the concatamer into smaller fragments comprising at least one delivery unit having a sequence of interest.
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Accused Products
Abstract
The invention provides an improved cell free amplification method capable of producing large quantities of therapeutic-quality nucleic acids and methods of using the synthesized nucleic acid in research, therapeutic and other applications—The methods combine several different state-of-the-art procedures and coordinate their applications to affordably synthesize nucleic acids for therapeutic purposes. It combines in vitro rolling circle amplification, high fidelity polymerases, high affinity primers, and streamlined template specifically designed for particular applications. For expression purposes, the templates contain an expression cassette including a eukaryotic promoter, the coding sequence for the gene of interest, and a eukaryotic termination sequence. Following amplification, concatamers are subsequently processed according to their intended use and may include: restriction enzyme digestion for the production of short expression cassettes (SECs); ligation steps to circularize the SEC (CNAs); and/or supercoiling steps to produce sCNAs. The final product contains nearly non-detectable levels of bacterial endotoxin.
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Citations
25 Claims
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1. A process for producing high-quality nucleic acid in a cell free system, comprising:
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(a) combining a circular template in a reaction mixture with one or more primers, which are complementary to at least one strand of the circular template, to form a template-primer complex; (b) incubating the template-primer complex with at least one high-fidelity nucleic acid polymerase to produce a concatamer comprising tandem units of the circular template; and (c) cutting the concatamer into smaller fragments comprising at least one delivery unit having a sequence of interest. - View Dependent Claims (2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14, 21, 22, 23, 24, 25)
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7. (canceled)
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13. (canceled)
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15. An apparatus for producing high-quality nucleic acid, comprising:
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a reaction vessel having at least one entry port and one exit port that before addition of reaction reagents, is clean, sterile and free of contaminating nucleic acid sequences; an input device attached to the entry port and which feeds at least one outside component from an outside holding chamber to the reaction vessel; at least one outside holding chamber connected to the input device; a means for pumping an outside component from the holding chamber, through the input device, into the reaction vessel; an export device attached to the exit port of the reaction vessel; at least one outside receiving chamber connected to the export device; a means for regulating temperature of the reaction vessel; a means for monitoring and controlling the progress of a reaction mixture within the reaction vessel; and a means for mixing the reaction mixture. - View Dependent Claims (16, 17)
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18. (canceled)
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19. (canceled)
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20. (canceled)
Specification