DNA sequencing reaction additive
The present invention provides methods, compositions, mixtures and kits utilizing 5-Chloro-2-methyl-4-isothiazolin-3-one in sequencing reactions, and in particular, sequencing reactions where deoxynucleoside triphosphates comprising a 3′-O position capped by a disulfide-based 3′-terminator group are used. In one embodiment, the deoxynucleoside triphosphates comprise 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 of incorporating nucleotides, comprising:
a) providing i) a plurality of nucleic acid primers and template molecules, and ii) an extend reagent comprising polymerase, a plurality of nucleotide analogues, wherein said nucleotide analogues comprise a 3′-O position capped by a disulfide-based 3′-terminator group and a label attached through a cleavable disulfide linker to the base, and 5-Chloro-2-methyl-4-isothiazolin-3-one;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; and
c) exposing said hybridized primers to said extend reagent in an extension reaction under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue, wherein 5-Chloro-2-methyl-4-isothiazolin-3-one inhibits cleavage of said disulfide-based 3′-terminator group during said extension reaction.
2. The method of claim 1 , wherein said disulfide-based 3′-terminator group comprises methylenedisulfide as a cleavable protecting group.
3. The method of claim 1 , wherein said extend reagent further comprises 2-Methyl-4-isothiazolin-3-one.
4. The method of claim 1 , further comprising:
d) detecting said label of a first labeled nucleotide analogue.
5. The method of claim 4 , further comprising:
e) cleaving said linker with a cleaving reagent so as to remove said label.
6. The method of claim 5 , wherein cleaving reagent also removes said disulfide-based 3′-terminator group.
7. The method of claim 6 , wherein said cleaving reagent is a thiol-containing compound.
8. The method of claim 7 , wherein the thiol-containing compound is a vicinal dithiol-based compound.
9. The method of claim 4 , wherein the vicinal dithiol-based compound is di-mercaptopropanesulfonate.
10. A method for detecting labeled nucleotides in a nucleic acid sequence comprising the steps of
a) providing i) nucleic acid template and primer capable of hybridizing to said template so as to form a primer/template hybridization complex, ii) an extend reagent comprising polymerase, a plurality of nucleotide analogues, and 5-Chloro-2-methyl-4-isothiazolin-3-one, wherein said nucleotide analogues comprise a nucleobase and a sugar, said sugar comprising a cleavable protecting group on the 3′-O, wherein said cleavable protecting group comprises a disulfide-based 3′-terminator group, and wherein at least a portion of said nucleotide analogues further comprises a first detectable label attached via a cleavable disulfide-containing linker to the nucleobase; and iii) a cleave reagent comprising a thiol-containing compound;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; and
c) exposing said hybridized primers to said extend reagent in an extension reaction under conditions such that a first nucleotide analogue comprising a first detectable label is incorporated into at least a portion of said hybridized primers in a polymerase catalyzed primer extension reaction so as to create extended primers comprising an incorporated nucleotide analogue in a modified primer/template hybridization complex, wherein 5-Chloro-2-methyl-4-isothiazolin-3-one inhibits cleavage of said disulfide-based 3′-terminator group during the extension reaction;
d) detecting said first detectable label of said deoxynucleoside triphosphate in said modified primer/template hybridization complex; 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.
11. The method of claim 10 , wherein the method further comprises incorporating a second nucleotide analogue comprising a second detectable label during a repeat of step c).
12. The method of claim 10 , wherein the thiol-containing compound is a vicinal dithiol-based compound.
13. The method of claim 12 , wherein the vicinal dithiol-based compound is di-mercaptopropanesulfonate.
14. The method of claim 11 , wherein the nucleobase of said second nucleotide analogue is different from the nucleobase of said first nucleotide analogue.
15. The method of claim 10 , wherein said extend reagent comprises a mixture of at least 4 differently labeled nucleotide analogues representing analogs of A, G, C and T or U.
16. The method of claim 10 , wherein said detecting allows for the determination of the nucleobase of said incorporated first nucleotide analogue.
17. The method of claim 10 , wherein said disulfide-based 3′-terminator group comprises methylenedisulfide as a cleavable protecting group.
18. The method of claim 10 , wherein said extend reagent further comprises 2-Methyl-4-isothiazolin-3-one.
19. The method of claim 10 , further comprising:
f) introducing a scavenger that interacts with the cleave reagent.
20. The method of claim 19 , wherein said scavenger is cystamine.