3′-OH unblocked nucleotides and nucleosides base modified with non-cleavable, terminating groups and methods for their use in DNA sequencing
Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a non-cleavable terminating group. The non-cleavable-fluorescent group is designed to terminate DNA synthesis so that DNA oligomers can be sequenced efficiently in a parallel format. These reagents and methods will lead to more accurate identification of polymorphisms and other valuable genetic information.
1. A compound selected from the group consisting of:
wherein R 1 ═H, monophosphate, diphosphate or triphosphate,
R 2 ═H or OH,
R 3 and R 4 are each independently selected from the group of H, a C 1 -C 12 straight chain or branched alkyl, a C 2 -C 12 straight chain or branched alkenyl or polyenyl, a C 2 -C 12 straight chain or branched alkynyl or polyalkynyl, and an aromatic group,
R 5 , R 6 , and R 7 , are each independently selected from the group consisting of H, OCH 3 , NO 2 , CN, a halide, a C 1 -C 12 straight chain or branched alkyl, a C 2 -C 12 straight chain or branched alkenyl or polyenyl, a C 2 -C 12 straight chain or branched alkynyl or polyalkynyl, and an aromatic group, with the proviso that one of R 5 , R 6 , and R 7 is a group of the structure —C≡CCH 2 NH 2 ,
wherein X═CH 2 , CH═CH, C≡C, O, S, or NH,
Y═CH 2 , O, or NH,
n=an integer from 0-12;
m=an integer from 0-12, and
Dye=a fluorophore,
and R 8 and R 9 are as defined above for R 5 , R 6 , and R 7 , with the proviso that R 8 and R 9 are not NO 2 .
2. A compound according to claim 1 , wherein R 3 and R 4 are each independently selected from the group consisting of H, —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , isopropyl, tert-butyl and phenyl.
3. The compound of claim 2 , wherein R 3 or R 4 is —CH 3 or isopropyl.
4. A compound according to claim 1 , wherein R 3 or R 4 are each independently selected from the group consisting of H, alkyl and aromatic groups optionally containing at least one heteroatom in the alkyl or aromatic groups, and further wherein the aromatic group may optionally be an aryl or polycyclic group.
5. The compound of claim 1 , further defined as:
6. The compound of claim 1 , further defined as:
7. The compound of claim 1 , further defined as:
8. The compound of claim 1 , further defined as:
9. The compound of claim 1 , further defined as:
10. The compound of claim 1 , further defined as a compound of formula (I).
11. The compound of claim 1 , further defined as a compound of formula (II).
12. The compound of claim 1 , further defined as a compound of formula (III).
13. The compound of claim 1 , further defined as a compound of formula (IV).
14. The compound of claim 1 , further defined as a compound of formula (V).
15. The compound of claim 1 , further defined as a compound of formula (VI).
16. The compound of claim 1 , further defined as a compound of formula (VII).