Nucleotide analogs
The invention provides nucleotide analogs for use in sequencing nucleic acid molecules.
1 . A labeled nucleotide analog of Formula I:
wherein,
R 1 at each occurrence, independently is selected from the group consisting of S, NR 3 and O,
R 2 is selected from the group consisting of H and OH,
R 3 is selected from the group consisting of H and alkyl,
R 5 is an aliphatic moiety,
B is selected from the group consisting of a purine, a pyrimidine, and analogs thereof,
L is a label, and
m is an integer from 1 to 3.
2 . The labeled nucleotide of claim 1 , wherein, in each occurrence, R 1 is S.
3 . The labeled nucleotide of claim 1 or 2 , wherein B is selected from the group consisting of cytosine, uracil, thymine, adenine, guanine, and analogs thereof.
4 . The labeled nucleotide analog of claim 1 , wherein L is an optically detectable label.
5 . The labeled nucleotide analog of claim 4 , wherein the optically detectable label is a fluorescent label.
6 . The labeled nucleotide analog of claim 4 , wherein the optically detectable label is selected from the group consisting of cyanine, rhodamine, fluoroscein, coumarin, BODIPY, alexa and conjugated multi-dyes.
7 . The labeled nucleotide analog of claim 4 , wherein the optically detectable label is Cy3 or Cy5.
8 . A method of removing a label or protecting group from a nucleotide, the method comprising the steps of:
(a) providing a nucleotide comprising a sugar and a label or protecting group linked via a phosphoryl moiety to a 3′ position of the sugar; and
(b) exposing the nucleotide to a reducing agent in an amount and under conditions to remove the label or protecting group.
9 . The method of claim 8 , wherein after step (b), the nucleotide comprises a phosphoryl group.
10 . The method of claim 9 , further comprising the step of exposing the nucleotide to a phosphatase to remove the phosphoryl moiety and produce a hydroxyl group.
11 . The method of claim 8 , wherein in step (b), the reducing agent is tris(2-chloroethyl) phosphate.
12 . A method of sequencing a nucleic acid template, the method comprising the steps of:
(a) exposing a nucleic acid template hybridized to a primer having a 3′ end to (i) a polymerase capable of catalyzing nucleotide additions to the primer, and (ii) the nucleotide analog of claim 1 under conditions to permit the polymerase to add the nucleotide analog to the 3′ end of the primer;
(b) detecting the nucleotide analog added to the primer in step (a); and
(c) removing the label from the nucleotide analog.
13 . The method of claim 12 , further comprising repeating steps (a), (b) and (c) thereby to determine the sequence of the template.
14 . The method of claim 12 , wherein, after step (c), the nucleotide analog has a hydroxyl group or the phosphoryl group.
15 . The method of claim 14 , wherein after step (c), the nucleotide analog is represented by formula II:
wherein,
R 2 is selected from the group consisting of H or OH,
R 4 is a phosphodiester linkage connecting the nucleotide analog to the primer, and
B is selected from the group consisting of a purine, a pyrimidine, and analogs thereof.
16 . The method of claim 15 , wherein, at step (c), the label is removed by exposure to a reducing agent.
17 . The method of claim 16 , where the reducing agent is tris(2-carboxyethyl) phosphine.
18 . The method of claim 16 , further comprising contacting the nucleotide analog with a phosphatase.