Thiol-containing cleave reagents and oxidative wash
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. In addition, thiol-containing compounds and scavengers of thio-containing compounds are described. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.
1. A method for detecting labeled nucleotides in a DNA sequence comprising the steps of
a) providing a nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, a thiol cleave reagent comprising di-mercaptopropanesulfonate, DNA polymerase, and 4 differently labeled populations of deoxynucleoside triphosphate compounds representing analogs of A, G, C and either T or U, wherein each of said labeled deoxynucleoside triphosphate compounds have a cleavable protecting group on the 3′-O and a detectable label linked via a cleavable linker, said compounds having the following structure:
wherein R 3 is selected from the group consisting of alkyl, substituted alkyl, allyl, and substituted allyl; B is a nucleobase; A is an attachment group; C is a cleavable site core selected from the group consisting of:
wherein R 1 and R 2 are independently selected alkyl groups, wherein L 1 and L 2 are connecting groups, 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;
b) adding said DNA polymerase and said labeled populations of deoxynucleoside triphosphate compounds to said primer and template so as to create a reaction mixture;
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, wherein a first labeled deoxynucleoside triphosphate is incorporated;
d) detecting said label of said first labeled deoxynucleoside triphosphate in said modified primer/template hybridization complex, wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate; and
e) introducing said cleave reagent under conditions so as to remove said cleavable protecting group and said detectable label from said modified primer/template hybridization complex, wherein the incidence of identifying the wrong base is reduced in comparison to other thiol cleave reagents.
2. The method of claim 1 , wherein the method further comprises repeating steps b)-e), wherein a second deoxynucleoside triphosphate is incorporated in step c).
3. A method for detecting labeled nucleotides in a DNA sequence comprising the steps of
a) providing a nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, a thiol cleave reagent comprising di-mercaptopropanesulfonate in a CHES buffer, wherein the pH of said cleave reagent is between 9.0 and 10.0, a cleave scavenger reagent comprising an oxidative scavenger, DNA polymerase, and 4 differently labeled populations of deoxynucleoside triphosphate compounds representing analogs of A, G, C and either T or U, wherein each of said labeled deoxynucleoside triphosphate compounds have a cleavable protecting group on the 3′-O and a detectable label linked via a cleavable linker, said compounds having the following structure:
wherein R 3 is selected from the group consisting of alkyl, substituted alkyl, allyl, and substituted allyl; B is a nucleobase; A is an attachment group; C is a cleavable site core selected from the group consisting of:
wherein R 1 and R 2 are independently selected alkyl groups, wherein L 1 and L 2 are connecting groups, 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;
b) adding said DNA polymerase and said labeled populations of deoxynucleoside triphosphate compounds to said primer and template so as to create a reaction mixture;
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, wherein a first labeled deoxynucleoside triphosphate is incorporated;
d) detecting said first detectable label of said first labeled deoxynucleoside triphosphate in said modified primer/template hybridization complex, wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate;
e) introducing said cleave reagent under conditions so as to remove said cleavable protecting group and said detectable label from said modified primer/template hybridization complex, wherein the incidence of identifying the wrong base is reduced in comparison to other thiol cleave reagents; and
f) introducing said cleave scavenger reagent.
4. The method of claim 3 , wherein said pH of said cleave reagent is 9.5.
5. The method of claim 3 , wherein said oxidative scavenger is hydrogen peroxide.
6. The method of claim 5 , wherein said hydrogen peroxide is in a buffer.
7. The method of claim 6 , wherein said buffer is TRIS.
8. The method of claim 7 , wherein said TRIS buffer is at a pH of between 8.5 and 9.0.
9. The method of claim 8 , wherein said TRIS buffer is at a pH of 8.8.
10. The method of claim 3 , wherein said oxidative scavenger is tert-butyl peroxide.
11. The method of claim 3 , wherein said reaction mixture of step b) is in a flow cell.
12. The method of claim 11 , wherein said flow cell is positioned on a moving support.
13. The method of claim 12 , wherein said moving support is a rotary stage.
14. The method of claim 11 , wherein at least a portion of said flow cell is transparent.
15. The method of claim 11 , wherein said flow cell is incorporated within an instrument.
16. The method according to claim 3 , wherein the method further comprises repeating steps b)-e), wherein a second deoxynucleoside triphosphate is incorporated in step c).
17. The method of claim 16 , wherein the nucleobase of said second deoxynucleoside triphosphate is different from the nucleobase of said first deoxynucleoside triphosphate.
18. A method for detecting labeled nucleotides in a DNA sequence comprising the steps of
a) providing nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, and a flow cell, said flow cell in fluidic communication with a first and second reservoir, said first reservoir comprising a thiol cleave reagent comprising di-mercaptopropanesulfonate in a CHES buffer, wherein the pH of said cleave reagent is between 9.0 and 10.0, said second reservoir comprising an oxidative wash, DNA polymerase, and 4 differently labeled populations of deoxynucleoside triphosphate compounds representing analogs of A, G, C and either T or U, wherein each of said labeled deoxynucleoside triphosphate compounds have a cleavable protecting group on the 3′-O and a detectable label linked via a cleavable linker, said compounds having the following structure:
wherein R 3 is selected from the group consisting of alkyl, substituted alkyl, allyl, and substituted allyl; B is a nucleobase; A is an attachment group; C is a cleavable site core selected from the group consisting of:
wherein R 1 and R 2 are independently selected alkyl groups, wherein L 1 and L 2 are connecting groups, 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;
b) adding said DNA polymerase and said labeled populations of deoxynucleoside triphosphate compounds to said primer and template so as to create a reaction mixture in said flow cell;
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, wherein a first labeled deoxynucleoside triphosphate is incorporated;
d) detecting said label of said first labeled deoxynucleoside triphosphate in said modified primer/template hybridization complex, wherein said detecting allows for the determination of the nucleobase of said incorporated first deoxynucleoside triphosphate;
e) introducing said cleave reagent from said first reservoir into said flow cell under conditions so as to remove said cleavable protecting group and said detectable label from said modified primer/template hybridization complex, wherein the incidence of identifying the wrong base is reduced in comparison to other thiol cleave reagents; and
f) introducing said oxidative wash from said second reservoir into said flow cell.
19. The method of claim 18 , wherein said pH of said cleave reagent is 9.5.
20. The method of claim 18 , wherein said oxidative wash comprises hydrogen peroxide.
21. The method of claim 20 , wherein said hydrogen peroxide is in a buffer.
22. The method of claim 21 , wherein said buffer is TRIS.
23. The method of claim 18 , wherein said oxidative wash comprises tert-butyl peroxide.
24. The method of claim 1 , wherein A is an attachment group selected from the group consisting of propargyl, a hydroxymethyl, an exocyclic amine, a propargyl amine, and a propargyl hydroxyl.
25. The method of claim 1 , wherein said Label is a label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes.
26. The method of claim 1 , 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—, CONH(CH 2 ) x , and —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from 0-10.
27. The method of claim 3 , wherein A is an attachment group selected from the group consisting of propargyl, a hydroxymethyl, an exocyclic amine, a propargyl amine, and a propargyl hydroxyl.
28. The method of claim 3 , wherein said Label is a label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes.
29. The method of claim 3 , 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—, CONH(CH 2 ) x —, and —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from 0-10.
30. The method of claim 18 , wherein A is an attachment group selected from the group consisting of propargyl, a hydroxymethyl, an exocyclic amine, a propargyl amine, and a propargyl hydroxyl.
31. The method of claim 18 , wherein said Label is a label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes.
32. The method of claim 18 , 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—, CONH(CH 2 ) x —, and —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from 0-10.