# US 2025/0084397 A1 · Methods of purifying dna for gene synthesis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0084397 A1 - Document ID: US20250084397A1 - Status: Published application - Kind: A1 - Application number: 18/725,999 - Filed: 2022-11-04 - Published: 2025-03-13 - Earliest claimed: 2022-01-04 - Assignees: ModernaTX, Inc. - Applicants: Modernatx, Inc. - Inventors: Kevin Smith - Web page: https://patentgrep.com/patent/US20250084397A1 ## CPC - C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) - C12N 15/1013: {by using magnetic beads} - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Provided herein are methods of purifying nucleic acids (e.g., DNA) for gene synthesis using combinations of nucleases. Also provided are improved products for use in the production of RNA. ## Claims ### Claim 1 A method for processing a DNA, the method comprising: preparing a sample of heteroduplex DNA, wherein at least one heteroduplex DNA in the sample comprises a mismatch DNA having one or more sequence errors, performing a dual nuclease digestion on the sample to produce a digested product by contacting the sample with T7E1 endonuclease to cleave the mismatch DNA at the sequence error site to produce one or more DNA fragments and contacting the sample with mung bean nuclease to degrade the DNA fragments, thereby producing a purified sample of heteroduplex DNA. ### Claim 2 (depends on 1) The method of claim 1, wherein the purified sample of heteroduplex DNA produced by the method has error-rate reductions of 15-60% relative to a comparable method performed without digestion with mung bean nuclease. ### Claim 3 (depends on 1) The method of claim 1, wherein the purified sample of heteroduplex DNA produced by the method has error-rate reductions of 20-30% relative to a comparable method performed without digestion with mung bean nuclease. ### Claim 4 (depends on 1, 2, 3) The method of any one of claims 1-3, wherein less than 5% of total nucleic acid in the purified sample of heteroduplex DNA is comprised of mismatched DNA and DNA fragments. ### Claim 5 (depends on 1, 2, 3, 4) The method of any one of claims 1-4, wherein at least 99% of heteroduplex DNA has 100% base complementarity and wherein at least 99% of the heteroduplex DNA is full length. ### Claim 6 (depends on 1, 2, 3, 4, 5) The method of any one of claims 1-5, wherein a re-assembly PCR step is performed following T7E1 endonuclease and mung bean nuclease digestion on the digested product, thereby producing a purified sample of DNA template. ### Claim 7 (depends on 6) The method of claim 6, wherein a purification step is performed following re-assembly PCR. ### Claim 8 (depends on 7) The method of claim 7, wherein the purification step is a solid-phase reversible immobilization (SPRI) paramagnetic bead process. ### Claim 9 (depends on 6, 7, 8) The method of any one of claims 6-8, wherein a purification step is not performed between the T7E1 endonuclease and mung bean nuclease digestion and the re-assembly PCR. ### Claim 10 (depends on 6, 7, 8, 9) The method of any one of claims 6-9, wherein the digested product is used in the re-assembly step at a maximum volume of 50 μL. ### Claim 11 The method of any one of the preceding claims, wherein the endonuclease is T7E1. ### Claim 12 The method of any one of the preceding claims, wherein the nuclease is wild type mung bean nuclease. ### Claim 13 The method of any one of the preceding claims, wherein the sample is contacted with the T7E1 and mung bean nucleases at the same time. ### Claim 13 The method of any one of the preceding claims, wherein the sample comprises 1:1 T7E1 endonuclease:mung bean nuclease. ### Claim 14 The method of any one of the preceding claims, wherein the sample is contacted with T7E1 and mung bean nucleases at separate times. ### Claim 14 The method of any one of the preceding claims, wherein the dual nuclease digestion step is performed at least two times. ### Claim 17 The method of any one of the preceding claims, wherein the process is a commercial batch process. ### Claim 18 The method of any one of the preceding claims, wherein the sequence error comprises a substitution, deletion, or insertion of between 1 and 10 nucleotides. ### Claim 19 The method of any one of the preceding claims, further comprising producing mRNA with the purified sample of heteroduplex DNA. ### Claim 20 A purified sample of DNA template, comprising a plurality of heteroduplex DNA, wherein at least 99% of the heteroduplex DNA has 100% base complementarity and wherein at least 99% of the heteroduplex DNA is full length. ### Claim 21 A purified sample of DNA template, comprising a plurality of DNA template, wherein at least 99% of the DNA template has 100% base complementarity and wherein at least 99% of the DNA template is full length. ### Claim 22 A composition comprising a heteroduplex DNA comprising a mismatch DNA having one or more sequence errors, T7E1 endonuclease, and mung bean nuclease. ### Claim 23 A composition comprising a plurality of heteroduplex DNA, T7E1 endonuclease, and mung bean nuclease, wherein at least 90-100% of the heteroduplex DNA is full length.