Molecules and methods for nucleic acid sequencing
The invention provides molecules and methods for nucleic acid synthesis reactions useful in sequencing-by-synthesis processes.
1 . A molecule of formula (I):
wherein,
Z is a purine, pyrimidine or analog thereof,
L is a linker;
Each F is independently an optically-detectable label;
R is alkyl; and
m is an integer greater than 1.
2 . The molecule of claim 1 , wherein the linker comprises an alkynyl group.
3 . The molecule of claim 1 , wherein the linker comprises the structure:
wherein,
n is an integer 1-7 inclusive; and
o is an integer 1-7 inclusive.
4 . The molecule of claim 1 , wherein each F is independently a fluorescent label.
5 . The molecule of claim 1 , wherein each F is independently cyanin-3 or cyanin-5.
6 . The molecule of claim 1 , wherein R is an alkyl having from about 1 to about 12 carbon atoms.
7 . The molecule of claim 1 , wherein the purine is adenine, guanine, or analog thereof.
8 . The molecule of claim 1 , wherein the pyrimidine is cytosine, thymidine, uracil, or analogs thereof.
9 . A molecule of formula (II):
wherein,
Z is a purine, pyrimidine or analog thereof,
L is a linker;
F is an optically-detectable label; and
m is an integer greater than 1.
10 . The molecule of claim 9 , wherein the linker comprises an alkynyl group.
11 . The molecule of claim 9 , wherein the linker comprises the structure:
wherein,
n is an integer 1-7 inclusive; and
o is an integer 1-7 inclusive.
12 . The molecule of claim 9 , wherein F is a fluorescent label.
13 . The molecule of claim 9 , wherein F is cyanin-3 or cyanin-5.
14 . The molecule of claim 9 , wherein the purine is adenine, guanine, or analog thereof.
15 . The molecule of claim 9 , wherein the pyrimidine is cytosine, thymidine, uracil, or analogs thereof.
16 . The molecule of claim 9 , wherein m is 2.
17 . The molecule of claim 1 , wherein m is 2.
18 . A method for sequencing a nucleic acid template comprising:
(a) exposing a nucleic acid duplex comprising a template nucleic acid hybridized to a primer nucleic acid to a plurality of molecules of a compound according to any of claims 1 - 17 under conditions that allow the molecule to be incorporated into the 3′-terminus of the primer and to engage in complementary base pairing with a nucleotide in the template.
19 . The method of claim 18 , further comprising:
(b) removing unincorporated molecules of the compound of any of claims 1 - 17 ; (c) observing a label associated with the compound of any of claims 1 - 17 ; (d) removing the label; (e) modifying the incorporated molecule to generate a free 3′-hydroxy group, and (f) repeating steps (a) to (e).
20 . The method of claim 19 , further comprising repeating step (f).
21 . The method of claim 19 , wherein step (b) comprises exposing the duplex to an agent capable of reducing disulfide bonds.
22 . The method of claim 18 , further comprising the step of identifying the molecule incorporated into the primer.
23 . The method of claim 19 , wherein step (d) comprises exposing the duplex to an agent capable of reducing disulfide bonds.
24 . The method of claim 19 , wherein step (e) comprises exposing the duplex to an agent capable of reducing disulfide bonds.
25 . The method of claim 19 , wherein steps (d) and (e) are performed simultaneously.
26 . The method of claim 21 , wherein the agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl).
27 . The method of claim 19 , wherein the reduction of the disulfide bond is performed at about pH 7.0 or greater.
28 . The method of claim 19 , wherein the reduction of the disulfide bond is performed at about pH 9.0 or greater.
29 . The method of claim 19 , wherein the reduction of the disulfide bond is performed at about 25° C. or greater.
30 . The method of claim 19 , wherein the reduction of the disulfide bond is performed at about 37° C. or greater.
31 . The method of claim 19 , wherein the reduction of the disulfide bond is performed at about 50° C. or greater.
32 . A method for synthesizing a nucleic acid analog comprising contacting a nucleic acid sequence with a compound of formula (I) in claim 1 or formula (II) in claim 9 .