Nucleotide analogues with 3'-pro-fluorescent fluorophores in nucleic acid sequence analysis
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Abstract
The invention concerns a novel class of 3′-modified, pro-fluorescent nucleotides. The invention also pertains to methods for using such nucleotides.
139 Citations
49 Claims
- 1. A compound of formula (I):
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6. A method for determining the identity of a nucleotide at at least one preselected site in a target polynucleotide, said method comprising the steps of:
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(a) incubating a target polynucleotide in the presence of at least one primer oligonucleotide, said primer having a sequence complementary to a sequence immediately 3′
to said preselected site of said target polynucleotide, said incubation being under conditions sufficient to permit said primer oligonucleotide to hybridize to said target oligonucleotide and to thereby form a hybridized product;
(b) further incubating said hybridized product in the presence of a mixture comprising a polymerase and at least one pro-fluorescent nucleotide species;
said incubation being under conditions sufficient to permit the polymerase-mediated template-dependent addition of said nucleotide species onto the 3′
-terminus of said hybridized primer oligonucleotide;
(c) permitting said polymerase to mediate the template-dependant addition of a pro-fluorescent nucleotide species onto the 3′
-terminus of said hybridized primer oligonucleotide, said addition being additionally dependant on said mixture containing a pro-fluorescent nucleotide species that is complementary to a nucleotide present at said preselected site;
(d) permitting the enzymatic hydrolysis of a reporter from said complementary pro-fluorescent nucleotide species; and
(e) determining the identity of said nucleotide at said preselected site from the identity of said reporter. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A method for determining the nucleic acid sequence of a target polynucleotide, said method comprising the steps of:
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(a) incubating a target polynucleotide in the presence of at least one primer oligonucleotide, said primer oligonucleotide having a sequence complementary to a sequence immediately 3′
to a first preselected site of said target polynucleotide, said incubation being under conditions sufficient to permit said primer oligonucleotide to hybridize to said target oligonucleotide and to thereby form a hybridized product;
(b) further incubating said hybridized product in the presence of a mixture comprising a polymerase and at least one terminator pro-fluorescent nucleotide species;
said incubation being under conditions sufficient to permit the polymerase-mediated template-dependant addition of said terminator pro-fluorescent nucleotide species onto the 3′
-terminus of said hybridized primer oligonucleotide;
(c) permitting said polymerase to mediate the template-dependant 3′
extension of said primer oligonucleotide by one terminator pro-fluorescent nucleotide species, said extension being additionally dependant on said mixture containing a terminator pro-fluorescent nucleotide species that is complementary to a nucleotide of the target polynucleotide present at said preselected site;
(d) permitting the enzymatic hydrolysis of a reporter from said complementary nucleotide species, thereby restoring a 3′
end suitable for the polymerase-mediated template-dependant extension of a said primer oligonucleotide by an additional terminator pro-fluorescent nucleotide species;
(e) determining the identity of said nucleotide at said preselected site from the identity of said hydrolyzed reporter;
(f) performing multiple iterations of steps (b) through (e), thereby in each iteration sequentially extending said primer oligonucleotide by one terminator pro-fluorescent nucleotide, and determining the identity of a next adjacent nucleotide of said target polynucleotide from the identity of said hydrolyzed reporter. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method for detecting a reporter comprising the steps of
(a) providing in a reaction a 3′ - -PF-ddNTP species and an enzyme, such that said 3′
-PF-ddNTP is utilized as a substrate in the reaction catalyzed by said enzyme, thereby hydrolyzing the reporter of said 3′
-PF-ddNTP species,said 3′
-PF-ddNTP species being of the formula N—
L1—
L2-Reporter, wherein N represents a nucleotide;
L1 represents at least one cleavable linking group selected from the group consisting of NH—
C(O)—
, NH—
C(S)—
, CH2CO, O—
C(O)—
, or —
OPO3—
, wherein one end of said cleavable linking group is attached to the 3′
position of said nucleotide;
L2 represents at least one spacer linking group selected from the group consisting of;
—
(NH—
CO)n—
or —
(OCH2—
CH2)n;
Reporter represents a chromophore or a pro-fluorescent fluorophore; and
(b) detecting said hydrolyzed reporter. - View Dependent Claims (47, 48)
- -PF-ddNTP species and an enzyme, such that said 3′
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49. A method for determining cell viability that comprises the steps of:
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(a) providing to a 3′
-PF-ddTNP species to a cell, such that hydrolysis of a reporter from said 3′
-PF-ddNTP is dependant upon the viability of said cell,said 3′
-PF-ddNTP species being of the formula N—
L1,—
L2-Reporter, wherein N represents a nucleotide;
L1 represents at least one cleavable linking group selected from the group consisting of NH—
C(O)—
, NH—
C(S)—
, CH2CO, O—
C(O)—
, or —
OPO3—
, wherein one end of said cleavable linking group is attached to the 3′
position of said nucleotide;
L2 represents at least one spacer linking group selected from the group consisting of;
—
(NH—
CO)n—
or —
(OCH2—
CH2)n;
Reporter represents a chromophore or a pro-fluorescent fluorophore; and
(b) detecting said hydrolyzed Reporter.
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Specification