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US 2026/0028616 A1Published application
Methods and apparatus for duplex nucleotide purification
AssigneeAgilent Technologies, Inc.
InventorsJoseph GUILES, Nevin GALLAGHER, Bob GRONEBERG
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Abstract
Disclosed herein are methods of purifying oligonucleotides, for example, by separating double-stranded oligonucleotides from single-stranded oligonucleotides. Also provided are apparatuses for performed such methods. Also provided are double-stranded nucleic acid compositions and single-stranded nucleic acid compositions made using the methods and apparatuses disclosed herein. Also provided are methods of producing circular double-stranded nucleic acids.
Claims20
A method of separating a double-stranded nucleic acid from a single-stranded nucleic acid, comprising:
- providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and
- subjecting the mixture to an ultra-filtration step that selectively retains double-stranded nucleic acid and not single-stranded nucleic acid,
- wherein the ultra-filtration step comprises applying the mixture to a filtration apparatus, wherein the filtration apparatus comprises a membrane with (i) a molecular weight (MW) cutoff that is at least 10% less than the MW of the double-stranded nucleic acid and at least 10% more than the MW of the single-stranded nucleic acid, and/or (ii) a pore size that will allow at least 90% of the single-stranded nucleic acids and at most 10% of the double-stranded nucleic acids to permeate the membrane,
- thereby separating a double-stranded nucleic acid from a single-stranded nucleic acid.
1 more element
An apparatus comprising:
- a first chamber configured to hold a mixture to be filtered, and optionally to capture retentate, a second chamber configured to capture permeate, and a filtration element disposed between the first and second chambers;
- wherein the mixture comprises double-stranded nucleic acid and single-stranded nucleic acid;
- wherein the filtration element selectively retains double-stranded nucleic acid and not single-stranded nucleic acid, optionally wherein the filtration element comprises a membrane with (i) a molecular weight (MW) cutoff that is at least 10% less than the MW of the double-stranded nucleic acid and at least 10% more than the MW of the single-stranded nucleic acid, and/or (ii) a pore size that will allow at least 90% of the single-stranded nucleic acids and at most 10% of the double-stranded nucleic acids to permeate the membrane.
A method of optimizing a technique for manufacturing a purified double-stranded nucleic acid, comprising:
- (i) providing a mixture comprising:
- a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid at a first concentration, and
- a single-stranded nucleic acid;
- wherein the mixture has a predetermined concentration of a salt;
- (ii) subjecting the mixture to an ultra-filtration step that selectively retains double-stranded nucleic acid comprising the first and second single-stranded nucleic acids and not the single-stranded nucleic acid, wherein the ultra-filtration step comprises applying the mixture to a filtration apparatus with a predetermined molecular weight cutoff for a predetermined length of time;
- (iii) harvesting the retentate;
- (iv) varying one of or more of the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined length of time, or the salt concentration;
- (v) repeating steps (i)-(iii) one or more times, each using one or more of the varied first concentration, second concentration, predetermined molecular weight cutoff, predetermined length of time, and/or salt concentration;
- (vi) determining, for each repetition of steps (i)-(iii), the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the harvested retentates;
- and
- (vii) identifying a first concentration, the second concentration, predetermined molecular weight cutoff, predetermined length of time, and/or salt concentration that yields the highest ratio of double-stranded nucleic acid to single-stranded nucleic acid in the harvested retentate;
- thereby optimizing a technique for manufacturing a purified double-stranded nucleic acid.
9 more elements
Dependencies are inferred from the claim text.