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US 2022/0186290 A1Published application
Compositions and methods for nucleotide modification-based depletion
AssigneeArc Bio, LLC
InventorStephane B. GOURGUECHON
Life of this patent
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Abstract
Provided herein are compositions and methods for enriching a sample for nucleic acids of interest relative to nucleic acids targeted for depletion, comprising using differences in nucleotide modification between the nucleic acids of interest and the nucleic acids targeted for depletion.
Claims70
A method of enriching a sample for nucleic acids of interest comprising:
- a. providing a sample comprising nucleic acids of interest and nucleic acids targeted for depletion, wherein at least a subset of the nucleic acids of interest or a subset of the nucleic acids targeted for depletion comprise a plurality of first recognition sites for a first modification-sensitive restriction enzyme;
- b. terminally dephosphorylating a plurality of the nucleic acids in the sample;
- c. contacting the sample from (b) with the first modification-sensitive restriction enzyme under conditions that allow for cleavage of at least some of the first modification-sensitive restriction sites in the nucleic acids in the sample;
- and
- d. contacting the sample from (c) with adapters under conditions that allow for the ligation of the adapters to a 5′ and 3′ end of a plurality of the nucleic acids of interest;
- thereby generating a sample enriched for nucleic acids of interest that are adapter-ligated on their 5′ and 3′ ends.
3 more elements
A method of enriching a sample for nucleic acids of interest comprising:
- a. providing a sample comprising nucleic acids of interest and nucleic acids targeted for depletion, wherein at least a subset of the nucleic acids targeted for depletion comprise a plurality of recognition sites for a modification-sensitive restriction enzyme;
- b. terminally dephosphorylating a plurality of the nucleic acids in the sample;
- c. contacting the sample from (b) with the modification-sensitive restriction enzyme under conditions that allow for the cleavage of the modification-sensitive restriction sites in the nucleic acids in the sample, thereby generating nucleic acids with exposed terminal phosphates;
- and
- d. contacting the sample with an exonuclease under conditions that allow for the successive removal of nucleotides from a phosphorylated end of a nucleic acid;
- thereby generating a sample enriched for nucleic acids of interest.
3 more elements
A method of enriching a sample for nucleic acids of interest comprising:
- a. providing a sample comprising nucleic acids of interest and nucleic acids targeted for depletion, wherein at least a subset of the nucleic acids targeted for depletion comprise a plurality of recognition sites for a modification-sensitive restriction enzyme;
- b. contacting the sample with adapters under conditions that allow for the ligation of the adapters to a 5′ and 3′ end of a plurality of the nucleic acids in the sample;
- and
- c. contacting the sample from (b) with the modification-sensitive restriction enzyme under conditions that allow for the cleavage of the modification-sensitive restriction sites in the nucleic acids in the sample;
- thereby generating a sample enriched for nucleic acids of interest that are adapter-ligated on their 5′ and 3′ ends.
2 more elements
A method of enriching a sample for nucleic acids of interest comprising:
- a. providing a sample comprising nucleic acids of interest and nucleic acids targeted for depletion,
- wherein at least a subset of the nucleic acids of interest or a subset of the nucleic acids targeted for depletion comprise a plurality of first recognition sites for a first modification-sensitive restriction enzyme, and
- wherein activity of the first modification-sensitive restriction enzyme is blocked by modification of a nucleotide within or adjacent to its cognate recognition site;
- b. terminally dephosphorylating a plurality of the nucleic acids in the sample;
- c. contacting the sample from (b) with the first modification-sensitive restriction enzyme under conditions that allow for cleavage of at least some of the first modification-sensitive restriction sites in the nucleic acids in the sample;
- and
- d. contacting the sample from (c) with adapters under conditions that allow for the ligation of the adapters to a 5′ and 3′ end of a plurality of the nucleic acids of interest;
- thereby generating a sample enriched for nucleic acids of interest that are adapter-ligated on their 5′ and 3′ ends.
5 more elements
Dependencies are inferred from the claim text.