Nucleotide analogues
Disclosed herein, interalia, are compounds, compositions, and methods of using the same for the sequencing of a nucleic acid.
1. A compound having the formula:
wherein,
B P is a nucleobase;
R 1P is hydrogen, or 5′-nucleoside protecting group, or —OR 1P is a monophosphate, or polyphosphate;
R 3P is hydrogen or —OR 3AP ; wherein R 3AP is hydrogen or a polymerase-compatible cleavable moiety;
L 1P is a substituted methylene, wherein L 1P is substituted with a haloalkyl, or substituted or unsubstituted heteroalkyl; and
R 6P is unsubstituted alkyl.
2. The compound of claim 1 , wherein R 3P is hydrogen or —OH.
3. The compound of claim 1 , wherein R 1P is hydrogen.
4. The compound of claim 1 , wherein OR 1P is a monophosphate moiety or polyphosphate moiety.
5. The compound of claim 1 , wherein B P is a monovalent cytosine or a derivative thereof, monovalent guanine or a derivative thereof, monovalent adenine or a derivative thereof, monovalent thymine or a derivative thereof, monovalent uracil or a derivative thereof, monovalent hypoxanthine or a derivative thereof, monovalent xanthine or a derivative thereof, monovalent 7-methylguanine or a derivative thereof, monovalent 5,6-dihydrouracil or a derivative thereof, monovalent 5-methylcytosine or a derivative thereof, or monovalent 5-hydroxymethylcytosine or a derivative thereof.
6. The compound of claim 1 , wherein B P is
7. The compound of claim 1 , wherein
B P is optionally substituted adenine, optionally substituted guanine, optionally substituted uracil, optionally substituted cytosine, optionally substituted thymine, optionally substituted hypoxanthine, optionally substituted xanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, or optionally substituted isoguanine.
8. The compound of claim 7 , wherein B P further comprises a fluorophore.
9. A method for sequencing a nucleic acid, comprising:
(i) incorporating in series with a nucleic acid polymerase, within a reaction vessel, one of four different compounds into a primer to create an extension strand, wherein said primer is hybridized to said nucleic acid and wherein each of the four different compounds comprises a unique detectable label;
(ii) detecting said unique detectable label of each incorporated compound, so as to thereby identify each incorporated compound in said extension strand, thereby sequencing the nucleic acid;
wherein each of said four different compounds is independently a compound of claim 1 .
10. A method of incorporating a compound into a primer, the method comprising combining a polymerase, a primer hybridized to nucleic acid template and the compound within a reaction vessel and allowing said polymerase to incorporate said compound into said primer thereby forming an extended primer, wherein said compound is a compound of claim 1 .
11. A compound having the formula:
wherein,
B P is a nucleobase;
R 1P is hydrogen, or 5′-nucleoside protecting group, or —OR 1P is a monophosphate, or polyphosphate;
R 3P is hydrogen or —OR 3AP ; wherein R 3AP is hydrogen or a polymerase-compatible cleavable moiety;
L 1P is a substituted methylene, wherein L 1P is substituted with substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; and
R 6P is unsubstituted C 2 alkyl.
12. A method for sequencing a nucleic acid, comprising:
(i) incorporating in series with a nucleic acid polymerase, within a reaction vessel, one of four different compounds into a primer to create an extension strand, wherein said primer is hybridized to said nucleic acid and wherein each of the four different compounds comprises a unique detectable label;
(ii) detecting said unique detectable label of each incorporated compound, so as to thereby identify each incorporated compound in said extension strand, thereby sequencing the nucleic acid;
wherein each of said four different compounds is independently a compound of claim 11 .
13. A method of incorporating a compound into a primer, the method comprising combining a polymerase, a primer hybridized to nucleic acid template and the compound within a reaction vessel and allowing said polymerase to incorporate said compound into said primer thereby forming an extended primer, wherein said compound is a compound of claim 11 .
14. The compound of claim 1 , wherein -L 1P -S—S—R 6P is:
15. The compound of claim 11 , wherein L 1P is substituted with unsubstituted alkyl or unsubstituted heteroalkyl.
16. The compound of claim 11 , wherein L 1P is substituted with unsubstituted alkyl.
17. The compound of claim 11 , wherein B P further comprises a fluorophore.
18. The compound of claim 11 , wherein -L 1P S—S—R 6P is: