Synthesis of DNA molecules in in vitro enzymatic systems

Patent metadata

Field	Value
Publication number	US-12371725-B2
Application number	US-202318494425-A
Country	US
Kind code	B2
Filing date	Oct 25, 2023
Priority date	Oct 25, 2022
Publication date	Jul 29, 2025
Grant date	Jul 29, 2025

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.

First claim

What is claimed is:

1. A method for synthesizing circular double-stranded DNA molecules, the method consisting of:
providing a DNA template comprising two sequence-specific recombination sites;
performing PCR to produce linear double-stranded DNA fragments comprising the two sequence-specific recombination sites;
converting the linear double-stranded DNA fragments to relaxed circular double-stranded DNA molecules via a recombination reaction in the presence of a recombinase;
removing unreacted linear double-stranded DNA fragments by adding an exonuclease; and
optionally, converting the relaxed circular double-stranded DNA molecules to supercoiled double-stranded DNA molecules.

2. The method of claim 1 , wherein the two sequence-specific recombination sites are selected from loxP sites, flippase recognition target (FRT) sites and a combination thereof.

3. The method of claim 1 , wherein the PCR is performed in the presence of a DNA polymerase, dNTPs and primers selected from sequences comprising SEQ ID NO: 1 or 2.

4. The method of claim 1 , wherein the recombinase is Cre recombinase.

5. The method of claim 1 , wherein the exonuclease is T5 exonuclease.

6. The method of claim 1 , wherein converting the relaxed circular double-stranded DNA molecules to supercoiled double-stranded DNA molecules comprises adding a DNA topoisomerase selected from DNA gyrase or DNA topoisomerase I.

7. The method of claim 1 , wherein the DNA template is a circular DNA template.

Assignees

• Leng Fenfei
• The Florida International Univ Board Of Trustees

Inventors

• Leng Fenfei

Classifications

• C12Q1/6846 Primary

  Common amplification features · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title