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US 2026/0049341 A1Published application
Method to generate supercoiled circular dna in vitro
AssigneeUniversity of Maryland, Baltimore County
InventorsCharles J. BIEBERICH, Xiang LI
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Abstract
Current minicircle production methods are slow, expensive, and difficult to perform under GMP conditions because, in most cases, the product is derived from bacteria. In contrast, HTLA-and CHTLA-based synthetic circular supercoiled DNA production can be done completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single stranded DNA, and/or double stranded DNA.
Claims20
A method of producing synthetic circular supercoiled DNA (SCSDNA), the method comprising:
- introducing at least two precursor DNA fragments into a buffer medium comprising a thermostable DNA ligase enzyme, wherein the precursor DNA fragments will correctly assemble to generate a defined DNA sequence;
- applying heat to a first temperature to cause the at least two precursor DNA fragments to denature;
- and
- lowering the temperature to a second temperature for:
- (i) annealing in the presence of the thermostable DNA ligase enzyme, thereby generating double-stranded DNA heteroduplexes formed by base pairing of complementary regions, a portion of the heteroduplexes having single-stranded 5′ overhangs and a portion of the heteroduplexes having 3′ overhangs, wherein when the 5′ intramolecular overhangs on a heteroduplex molecule are complementary and ligation on both DNA strands occurs, SCSDNA is produced, and when the 3′ intramolecular overhangs on a heteroduplex molecule are complementary and ligation on both DNA strands occurs, SCSDNA is produced;
- and (ii) substantial supercoiling of the SCSDNA in the presence of the thermostable type II topoisomerase.
3 more elements
Dependencies are inferred from the claim text.