Methods of purifying dna for gene synthesis
AssigneeModernaTX, Inc.
InventorKevin Smith
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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.
Claims23
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.
The method of any one of the preceding claims, wherein the endonuclease is T7E1.
The method of any one of the preceding claims, wherein the nuclease is wild type mung bean nuclease.
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.
The method of any one of the preceding claims, wherein the sample comprises 1:1 T7E1 endonuclease:mung bean nuclease.
The method of any one of the preceding claims, wherein the sample is contacted with T7E1 and mung bean nucleases at separate times.
The method of any one of the preceding claims, wherein the dual nuclease digestion step is performed at least two times.
The method of any one of the preceding claims, wherein the process is a commercial batch process.
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.
The method of any one of the preceding claims, further comprising producing mRNA with the purified sample of heteroduplex DNA.
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.
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.
A composition comprising a heteroduplex DNA comprising a mismatch DNA having one or more sequence errors, T7E1 endonuclease, and mung bean nuclease.
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.
Dependencies are inferred from the claim text.