Synthesis of dna molecules in in vitro enzymatic systems
AssigneeThe Florida International University Board of Trustees
InventorFenfei LENG
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Abstract
A method, which synthesizes closed circular single-stranded and double-stranded DNA molecules using in vitro enzymatic systems, is described. Circular single-stranded DNA molecules and double-stranded DNA molecules (e.g., relaxed, or supercoiled) with various sizes can be synthesized. Unwanted DNA molecules, e.g., unligated oligomers, can be removed by exonucleases, such as T5 exonuclease, T7 exonuclease, lambda exonuclease, E. coli exonuclease I and/or III. A method of converting the single-stranded circular DNA molecules into double-stranded circular DNA molecules is also described. The single-stranded and double-stranded circular DNA molecules can be used in a variety of applications.
Claims13
A method for synthesizing a circular double-stranded DNA molecule, the method comprising:
- providing a nicked circular DNA template comprising two sequence-specific recombination sites;
- performing rolling circle amplification (RCA) to produce a double-stranded DNA product comprising the two sequence-specific recombination sites;
- adding an endonuclease to the double-stranded DNA product to produce a linear double-stranded DNA fragment comprising the two sequence-specific recombination sites;
- converting the linear double-stranded DNA fragment to a relaxed circular double-stranded DNA molecule;
- and
- optionally, converting the relaxed circular double-stranded DNA molecule to a supercoiled double-stranded DNA molecule.
A kit comprising a DNA template comprising two sequence-specific recombination sites, DNA primers, a DNA polymerase, dNTPs, a recombinase, DNA Topoisomerase, buffers, T5 exonuclease, and an endonuclease.
Dependencies are inferred from the claim text.