Chemically Cleavable 3'-O-Allyl-DNTP-Allyl-Fluorophore Fluorescent Nucleotide Analogues and Related Methods
First Claim
1. A nucleotide analogue comprising (i) a base selected from the group consisting of adenine or an analogue of adenine, guanine or an analogue of guanine, cytosine or an analogue of cytosine, thymine or an analogue of thymine and uracil or an analogue of uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′
- -oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker.
3 Assignments
0 Petitions
Accused Products
Abstract
This invention provides a nucleotide analogue comprising (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine and uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker, and methods of nucleic acid sequencing employing the nucleotide analogue.
-
Citations
32 Claims
-
1. A nucleotide analogue comprising (i) a base selected from the group consisting of adenine or an analogue of adenine, guanine or an analogue of guanine, cytosine or an analogue of cytosine, thymine or an analogue of thymine and uracil or an analogue of uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′
- -oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker.
- View Dependent Claims (2, 3, 4, 5, 6)
-
7. A method for making a nucleotide analogue wherein the nucleotide analogue comprises (i) a base selected from the group consisting of adenine or an analogue of adenine, guanine or an analogue of guanine, cytosine or an analogue of cytosine and uracil or an analogue of uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′
- oxygen of the deoxyribose, and (iv) a fluorophore bound to the base via an allyl linker which is not an iso-allyl linker, comprising the steps of;
(a) contacting 6-amino-hex-2-en-1-ol and an N-hydroxysuccinimide ester of a fluorophore in the presence of a first suitable solvent and a suitable base; (b) treating the resulting product of step (a) with DSC/Et3N in a second suitable solvent; and (c) treating the resulting product of step (b) with a 3′
-O-allyl-dNTP-NH2 in the presence of a suitable buffered solvent, wherein the base of the 3′
-O-allyl-dNTP-NH2 is an adenine, guanine, cytosine, uracil, or an analogue thereof, thereby making the nucleotide analogue. - View Dependent Claims (8, 9, 10, 11, 12, 13)
- oxygen of the deoxyribose, and (iv) a fluorophore bound to the base via an allyl linker which is not an iso-allyl linker, comprising the steps of;
-
14. A method for making a nucleotide analogue wherein the nucleotide analogue comprises (i) a base selected from the group consisting of adenine or an analogue of adenine, guanine or an analogue of guanine, cytosine or an analogue of cytosine and uracil or an analogue of uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′
- oxygen of the deoxyribose, and (iv) a fluorophore bound to the base via an allyl linker, comprising the steps of;
(a) contacting 2-(2-amino-ethyl)-prop-2-en-1-ol and an N-hydroxysuccinimide ester of a fluorophore in the presence of a first suitable solvent and a suitable base; (b) treating the resulting product of step (a) with DSC/Et3N in a second suitable solvent; and (c) treating the resulting product of step (b) with a 3′
-O-allyl-dNTP-NH2 in the presence of a suitable buffered solvent, wherein the base of the 3′
-O-allyl-dNTP-NH2 is an adenine, guanine, cytosine, uracil, or an analogue thereof, thereby making the nucleotide analogue. - View Dependent Claims (15, 16, 17, 18)
- oxygen of the deoxyribose, and (iv) a fluorophore bound to the base via an allyl linker, comprising the steps of;
-
19. A method for determining the sequence of a DNA comprising performing the following steps for each residue of the DNA to be sequenced:
-
(a) contacting the DNA with a DNA polymerase in the presence of (i) a primer and (ii) four fluorescent nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (1) the nucleotide analogues consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of DATP, (2) each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′
-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker, so that a nucleotide analogue complementary to the residue being sequenced is bound to the DNA by the DNA polymerase, and (3) each of the four analogues has a predetermined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;(b) removing unbound nucleotide analogues; (c) determining the identity of the bound nucleotide analogue; and (d) following step (c), except with respect to the final DNA residue to be sequenced, chemically cleaving from the bound nucleotide analogue the fluorophore and the allyl moiety bound to the 3′
-oxygen atom of the deoxyribose,thereby determining the sequence of the DNA. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
-
- 31. A method for covalently affixing a detectable moiety, via an allyl linker, to an NH2-bearing molecule, comprising contacting the detectable moiety with the NH2-bearing molecule in the presence of a suitable solvent and suitable base, wherein the detectable moiety comprises a mass tag, fluorophore or chromophore bound to a NHS ester of an allyl moiety.
Specification