Methods of using nucleotide analogues
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.
1. 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 R 1 and R 2 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.
2. The method according to claim 1 , wherein said DNA polymerase catalyzed primer extension reaction is part of a sequencing reaction.
3. 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 R 1 and R 2 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.
4. The method according to claim 3 , further comprising the steps of e) removing said cleavable protecting group, and f) repeating steps b) to e) at least once.
5. The method according to claim 4 , wherein the method further comprises adding a second deoxynucleoside triphosphate during repeat of step b), wherein said second deoxynucleoside triphosphate comprises a second detectable label attached via a cleavable oxymethylenedisulfide linker, wherein said second detectible label is different from said first detectible label.
6. The method according to claim 5 , wherein the nucleobase of said second deoxynucleoside triphosphate is different from the nucleobase of said first deoxynucleoside triphosphate.
7. The method according to claim 4 , wherein a mixture of at least 4 differently labeled, 3′-O methylenedisulfide capped deoxynucleoside triphosphate compounds representing analogs of Adenosine, Guanosine, Cytidine, and Thymidine or Uridine are used in step b).
8. The method according to claim 4 , wherein step e) is performed by exposing said modified primer/template hybridization complex to a reducing agent.
9. The method according to claim 8 , wherein said reducing agent is tris(2-carboxyethyl)phosphine.
10. The method according to claim 4 , wherein step e) is performed by exposing said modified primer/template hybridization complex to a thiol-containing compound.
11. The method according to claim 3 , wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate.
12. The method according to claim 4 , wherein said detectable label from said modified primer/template hybridization complex is removed prior to step e).
13. 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 R 1 and R 2 are independently selected alkyl groups; L 1 and L 2 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.
14. The method according to claim 13 , wherein said DNA polymerase catalyzed primer extension reaction is part of a sequencing reaction.
15. The method according to claim 13 , wherein L 1 is selected from the group consisting of —CONH(CH 2 ) x —, —CO—O(CH 2 ) x —, —CONH—(OCH 2 CH 2 O) x —, —CO—O(CH 2 CH 2 O) x —, and —CO(CH 2 ) x —, wherein x is 0-10.
16. The method according to claim 13 , wherein L 2 is selected from the group consisting of
—NH—, —(CH 2 ) x —NH—, —C(Me) 2 (CH 2 ) x NH—, —CH(Me)(CH 2 ) x NH—, —C(Me) 2 (CH 2 ) x CO—, —CH(Me)(CH 2 ) x CO—, —(CH 2 ) x OCONH(CH 2 ) y O(CH 2 ) z NH—, —(CH 2 ) x CONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —(CH 2 ) x OCONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —CONH(CH 2 ) x —, and —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from is 0-10.
17. 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 R 1 and R 2 are independently selected alkyl groups; L 1 and L 2 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.
18. The method according to claim 17 , wherein L 1 is selected from the group consisting of —CONH(CH 2 ) x —, —CO—O(CH 2 ) x —, —CONH—(OCH 2 CH 2 O) x —, —CO—O(CH 2 CH 2 O) x —, and —CO(CH 2 ) x —, wherein x is 0-10.
19. The method according to claim 17 , wherein L 2 is selected from the group consisting of
—NH—, —(CH 2 ) x —NH—, —C(Me) 2 (CH 2 ) x NH—, —CH(Me)(CH 2 ) x NH—, —C(Me) 2 (CH 2 ) x CO—, —CH(Me)(CH 2 ) x CO—, —(CH 2 ) x OCONH(CH 2 ) y O(CH 2 ) z NH—, —(CH 2 ) x CONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —(CH 2 ) x OCONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —CONH(CH 2 ) x —, and —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from is 0-10.