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US 10,273,539 B2Granted
Methods of using nucleotide analogues
AssigneeQiagen Sciences, LLC
InventorsMong Sano Marma, Jerzy Olejnik
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Abstract
The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.
Claims19
A method of performing a DNA synthesis reaction comprising the steps of
- a) providing a nucleic acid template with a primer hybridized to said template, a DNA polymerase, at least one deoxynucleoside triphosphate having the structure:
- wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups;
- B is a nucleobase;
- Linker comprises a cleavable oxymethylenedisulfide-containing site core, wherein said cleavable site core is selected from the group consisting of:
- wherein R1 and R2 are independently selected alkyl groups;
- and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes, and
- b) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction.
4 more elements
A method for analyzing a DNA sequence comprising the steps of
- a) providing a nucleic acid template with a primer hybridized to said template forming a primer/template hybridization complex,
- b) adding DNA polymerase, and a first deoxynucleoside triphosphate having the structure:
- wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups;
- B is a nucleobase;
- Linker comprises a cleavable oxymethylenedisulfide-containing site core, wherein said cleavable site core is selected from the group consisting of:
- wherein R1 and R2 are independently selected alkyl groups;
- and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes,
- c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, and
- d) detecting said first detectable label of said deoxynucleoside triphosphate in said modified primer/template hybridization complex.
6 more elements
A method of performing a DNA synthesis reaction comprising the steps of
- a) providing a nucleic acid template with a primer hybridized to said template, a DNA polymerase, at least one deoxynucleoside triphosphate having the structure:
- wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups;
- B is a nucleobase;
- A is an attachment group selected from the group consisting of propargyl, exocyclic amine, propargyl amine, and propargyl hydroxyl;
- C is a cleavable site core selected from the group consisting of:
- wherein R1 and R2 are independently selected alkyl groups;
- L1 and L2 are connecting groups;
- and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes, and
- b) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction.
6 more elements
A method for analyzing a DNA sequence comprising the steps of
- a) providing a nucleic acid template with a primer hybridized to said template forming a primer/template hybridization complex,
- b) adding DNA polymerase, and a first deoxynucleoside triphosphate having the structure:
- wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups;
- B is a nucleobase;
- A is an attachment group selected from the group consisting of propargyl, exocyclic amine, propargyl amine, and propargyl hydroxyl;
- C is a cleavable site core selected from the group consisting of:
- wherein R1 and R2 are independently selected alkyl groups;
- L1 and L2 are connecting groups;
- and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes,
- c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, and
- d) detecting said first detectable label of said deoxynucleoside triphosphate in said modified primer/template hybridization complex.
8 more elements
Dependencies are inferred from the claim text.