US2003055257A1PendingUtilityA1
Water-soluble rhodamine dye conjugates
Est. expiryNov 3, 2019(expired)· nominal 20-yr term from priority
G01N 2458/00C07H 21/00C09B 11/24C07D 311/80G01N 33/582C07D 491/04C07D 491/06Y10S436/80C07H 19/207
48
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Claims
Abstract
The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A rhodamine dye or a salt thereof, comprising a rhodamine-type parent xanthene ring having attached to the xanthene C9 carbon a phenyl group that is further substituted with an ortho carboxy or ortho sulfonate group or a salt thereof, one to three substituted or unsubstituted aminopyridimnum groups and a substituted or unsubstituted alkylthio, arylthio or heteroarylthio group, said rhodamine dye optionally including one or more linking moieties.
2 . The rhodamine dye of claim 1 which comprises the structure:
wherein:
n is 1, 2, or 3;
Y is a rhodamine-type parent xanthene ring attached to the illustrated phenyl group at the xanthene C9 carbon;
each R is independently selected from the group consisting of (C 1 -C 6 ) alkyl and heteroalkl, (C 1 -C 20 ) aryl and heteroaryl (C 6 -C 26 ) arylalkyl and heteroalkyl, (C 5 -C 20 ) arylaryl and heteroaryl-heteroaryl, or when taken together, R is (C 4 -C 10 ) alkyldiyl, (C 4 -C 10 ) alkyleno, heteroalkyldiyl and heteroalkyleno;
S is sulfuir;
Z is (C 1 -C 12 ) alkyl, (C 1 -C 12 ) alkyl substituted with one or more of the same or different W 1 groups, (C 5 -C 20 ) aryl and heteroaryl, and (C 5 -C 20 ) aryl and heteroaryl substituted with one or more of the same or different W 2 groups;
W 1 is selected from the group consisting of —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX 3 , —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO 2 , ═N 2 , —N 3 , —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR;
W 2 is selected from the group consisting of —R, —OR, —SR, —NRR, —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)OF, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR;
each X is independently a halogen; and
Y or Z is optionally substituted with L where L is a bond or a linker.
3 . The rhodamine dye of claim 2 in which L is selected from a hydrophobic moiety, a charged group, a member of a pair of specific binding molecules, a photo-activatable group and a reactive functional group.
4 . The rhodamine dye of claim 2 where Z has the form Z 1 —L—R X , or a salt thereof, wherein:
Z 1 is (C 1 -C 12 ) alkyldiyl, (C 1 -C 12 ) alkyldiyl independently substituted with one or more of the same or different W 1 groups, or
(C 5 -C 14 ) aryldiyl, and aryldiyl, heteroaryldiyl and heteroaryldiyl independently substituted with one or more of the same or different W 2 groups;
L is a bond or a linker; and
R X is a reactive functional group.
5 . The rhodamine dye of claim 4 in which Y is selected from:
and a salt thereof, wherein:
R 1 and le When taken alone, are independently hydrogen or (C 1 -C 6 ) alkyl;
R 3 and R 3′ when taken alone, are independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 14 ) aryl and arylaryl, or when taken together is (C 4 -C 6 ) alkyidyl or alkyleno, or when individually taken together with R 2 or R 4 is (C 2 -C 6 ) alkyldiyl or (C 2 -C 6 ) alkyleno;
R 4 , when taken alone, is selected from the group consisting of hydrogen and (C 1 -C 6 ) alkyl, or when taken together with R 3 or R 3′ is (C 2 -C 6 ) alkyldiyl or alkyleno;
R 5 , when taken alone, is selected from the group consisting of hydrogen and (C 1 -C 6 ) alkyl, or when taken together with R 6 or R 6′ is (C 2 -C 6 ) alkyldiyl or alkyleno;
R 6 and R 6′ when taken alone, are selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl and arylaryl, or when taken together are (C 4 -C 6 ) alkyldiyl or alkyleno, or when individually taken together with R 5 or R 7 is (C 2 -C 6 ) alkyldiyl or alkyleno;
R 7 , when taken alone, is selected from the group consisting of hydrogen and (C 1 -C 6 ) alkyl, or when taken together with le or R 6 is (C 2 -C 6 ) alkyldiyl or alkyleno;
R 8 , when taken alone, is selected from the group consisting of hydrogen and (C 1 -C 6 ) alkyl;
R 9 indicates the point of attachment to the ortho-carboxyphenyl bottom ring; and
R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are each independently selected from the group consisting of hydrogen and (C 1 -C 6 ) alkyl, or
when R 10 , R 11 , R 12 and R 13 taken together are (C 5 -C 14 ) aryleno or (C 5 -C 14 ) aryleno substituted with one or more of the same or different (C 1 -C 6 ) alkyl, or
when R 18 , R 19 , R 20 and R 21 taken together are (C 5 -C 14 ) aryleno or aryleno substituted with one or more of the same or different (C 1 -C 6 ) alkyl.
6 . The rhodamine dye of claim 5 wherein R 2 , when taken together with R 3 or R 3′ is (C 2 -C 6 ) alkyldiyl or alkyleno.
7 . The rhodamine dye of claim 6 wherein:
alkyl is methanyl, ethanyl or propanyl;
aryl is phenyl or naphthyl;
arylaryl is biphenyl;
alkyldiyl or alkyleno bridges formed by taking R 2 together with R 3 or R 3′ , R 7 together with R 6 or R 6′ , or R 4 together with and R 3 or R 3′ , are ethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano;
aryleno bridges formed by taking R 1 together with R 2 are benzo or naphtho;
alkyldiyl or alkyleno bridge formed by taking R 3 together with R 3′ , or R 6 together with R 6 , is butano;
alkyldiyl or alkyleno bridges formed by taking R 5 together with R 6 or R 6′ are ethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano; and
aryleno bridge formed by taking R 10 , R 11 , R 12 and R 13 together, or R 18 , R 19 , R 20 and R 21 together, is benzo.
8 . The rhodamine dye of claim 6 in which L is a bond.
9 . The rhodamine dye of claim 4 in which R X is selected from the group consisting of carboxyl, carboxylate, ester and activated ester.
10 . The rhodamine dye of claim 4 in which Z 1 is selected from the group consisting of (C 1 -C 12 ) alkyleno, (C 1 -C 12 ) alkano, (C 5 -C 10 ) aryldiyl and heteroaryldiyl, phenyldiyl, phena-1,4-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyl and purindiyl.
11 . The rhodamine dye of claim 4 in which Y is selected from the group consisting of:
12 . The rhodamine dye of claim 4 in which L is a bond.
13 . The rhodamine dye of claim 4 in which R X is selected from the group consisting of carboxyl, carboxylate, ester and activated ester.
14 . The rhodamine dye of claim 4 in which Z 1 is selected from the group consisting of (C 1 -C 12 ) alkyleno, (C 1 -C 12 ) alkano, (C 5 -C 10 ) aryldiyl and heteroarylidyl, phenylidyl, phena-14-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyl and purindiyl.
15 . The rhodamine dye of claim 4 which comprises the structure:
or a salt thereof.
16 . The rhodamine dye of claim 15 in which Y is selected from the group consisting of Y-1, Y-2, Y-3 and Y-4.
17 . The rhodamine dye of claim 15 in which Y is selected from the group consisting of Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
18 . The rhodamine dye of claim 2 which has the structure:
wherein:
Y 1 is a rhodamine-type parent xanthene ring attach ed to the illustrated phenyl group at the xanthene C9 carbon;
L is a bond or linker attached to a xanthene nitrogen atom or a xanthene C4 carbon;
n is 1, 2, or 3; and
R X is a reactive functional group.
19 . The rhodamine dye of claim 18 in which Y 1 is selected from the group consisting of:
wherein the dashed line at the nitrogen or C4 atom indicates the point of attachment of substituent L.
20 . The rhodamine dye of claim 19 wherein:
alkyl is methanyl, ethanyl or propanyl;
aryl is phenyl or naphthyl;
arylaryl is biphenyl;
alkyldiyl or alkyleno bridges formed by taking R 2 together with R 3 , R 4 together with R 3′ , R 5 together with R 6 , or R 7 together with R 6′ , are ethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano;
aryleno bridges formed by taking R 10 , R 11 , R 12 and R 13 together or R 18 , R 19 , R 20 and R 21 together are benzo.
21 . The rhodamine dye of claim 18 in which L is selected from the group consisting of (C 1 -C 6 ) alkyldiyl, (C 1 -C 6 ) alkano, (C 5 -C 20 ) aryldiyl, phenyldiyl, phena-1,4-diyl, naphthyldiyl, naphtha-2,6-diyl, naphtha-2,7-diyl, (C 6 -C 26 ) arylalkyldiyl —(CH) i -Φ- and —(CH) i -ψ-, where each i is independently an integer from 1 to 6, φ is (C 5 -C 20 ) aryldiyl, phenyldiyl or phena-1,4-diyl and ψ is naphthyldiyl, naphtha-2,6-diyl or naphtha-2,7-diyl.
22 . The rhodamine dye of claim 18 in which R X is selected from the group consisting of carboxyl, carboxylate, ester and activated ester.
23 . The rhodamine dye of claim 18 in which Z is selected from the group consisting of (C 1 -C 12 ) alkyl, (C 1 -C 12 ) alkanyl, (C 5 -C 10 ) aryl and heteroaryl, phenyl, naphthyl, naphth-1-yl, naphth-2-yl, pyridyl and purinyl.
24 . The rhodamine dye of claim 18 in which Y 1 is selected from the group consisting of:
wherein R 9 and the dash at the nitrogen or C4 atom indicates the point of attachment of L.
25 . The rhodamine dye of claim 18 which has the structure:
26 . The rhodamine dye of claim 25 in which Y 1 is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4b, Y-1c, Y-2c, Y-3c and Y-4c.
27 . The rhodamine dye of claim 25 in which Y 1 is selected from the group consisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-3b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.
28 . An energy-transfer dye pair comprising a donor dye linked to an acceptor dye, wherein the donor dye or the acceptor dye is a compound according to claim 1 and either or both of said donor and acceptor dyes include an optional linking moiety.
29 . The dye pair of claim 28 which has the structure:
or a salt thereof, wherein:
R 41 is a covalent linkage formed upon reaction between a nucleophile and an electrophile;
L″ is a bond or a linker;
n is 1,2, or 3; and
DD/AD is a donor dye or an acceptor dye which includes a linking moiety.
30 . The dye pair of claim 29 in which Y is selected from the group consisting of Y-1, Y-2, Y-3, Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31 a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
31 . The dye pair of claim 29 in which L is a bond.
32 . The dye pair of claim 29 in which R 41 has the formula —C(O)NR 45 —, where R 45 is hydrogen or (C 1 -C 6 ) alkyl.
33 . The dye pair of claim 29 in which Z 1 is selected from the group consisting of (C 1 -C 12 ) alkyleno, (C 1 -C 12 ) alkano, (C 5 -C 10 ) aryldiyl and heteroaryldiyl, phenyldiyl, phena-1,4-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyl and purindiyl.
34 . The dye pair of claim 29 in which L″ is —R 43 —Z 3 —C(O)—R 44 —R 45 —, wherein R 43 is (C 1 -C 6 ) alkyldiyl, preferably (C 1 -C 3 ) alkano, and is bonded to R 42 , where R 42 is O, S or NH; Z 3 is 5-6 membered cyclic alkenyldiyl and heteroalkenyldiyl, (C 1 -C14) aryldiyl and heteroaryldiyl; R 44 is O, S or NH; and R 45 is (C 1 -C 6 ) alkyldiyl, preferably (C 1 -C 3 ) alkano.
35 . The dye pair of claim 29 in which DD/AD is a fluorescein dye in which the linking moiety is a reactive functional group and wherein L″ is attached to the fluorescein dye at the xanthene C4 carbon.
36 . The dye pair of claim 29 which has the structure:
wherein, R 50 is a carboxyl, a salt, ester or activated ester thereof.
37 . The dye pair of claim 36 in which Y is selected from the group consisting of Y-1, Y-2, Y-3, Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41 a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
38 . The dye pair of claim 28 which has the structure:
wherein:
R 41 is a covalent linkage formed upon reaction between a nucleophile and an electrophile;
L″ is a bond or a linker;
n is 1, 2, or 3; and
DD/AD is a donor dye or an acceptor dye which includes a linking moiety.
39 . The dye pair of claim 38 in which Y 1 is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4b, Y-1c, Y-2c, Y-3c, Y-4c, Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.
40 . The dye pair of claim 38 in which L is (C 1 -C 6 ) alkyldiyl or (C 1 -C 3 ) alkano.
41 . The dye pair of claim 38 in which R 41 is an amide of the formula —C(O)NR 45 , where R 45 is hydrogen or (C 1 -C 6 ) alkyl.
42 . The dye pair of claim 38 in which Z is selected from the group consisting of (C 1 -C 12 ) alkyl, (C 1 -C 12 ) alkanyl, (C 5 -C 10 ) aryl and heteroaryl, phenyl, naphthyl, naphth-1yl, naphth-2-yl, pyridyl and purinyl.
43 . The dye pair of claim 38 in which L″ is —R 43 —Z 3 —C(O)—R 44 —R 45 —, wherein R 43 is (C 1 -C 6 ) alkydiyl, preferably (C 1 -C 3 ) alkano, and is bonded to R 42 , where R 42 is O, S or NH; Z 3 is 5-6 membered cyclic alkenyldiyl and heteroalkenyldiyl, (C 5 -C 14 ) aryldiyl and heteroaryldiyl; R 44 is O, S or NH; and R 45 is (C 1 -C 6 ) alkyldiyl, preferably (C 1 -C 3 ) alkano.
44 . The dye pair of claim 38 in which DD/AD is a fluorescein dye in which the linking moiety is a reactive group R X and wherein L″ is attached to the fluorescein dye at the xanthene C5 carbon.
45 . The dye pair of claim 38 which has the structure:
wherein:
Y 1 is selected from the group consisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c; and
R 50 is a carboxyl, a salt, ester or activated ester thereof.
46 . The dye pair of claim 45 in which Y 1 is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4b, Y-1c, Y-2c, Y-3c, Y-4c, Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.
47 . A labeled nucleoside/tide or nucleoside/tide analog comprising the rhodamine dye of claim 2 where Z has the form Z 1 —L—R 46 —L′—NUC, wherein:
R 46 is a linkage formed by reaction between an electrophile and a nucleophile; and —L′—NUC taken together has the structure:
wherein:
B is a nucleobase;
L′ is (C 1 -C 20 ) alkyldiyl and heteroalkyldiyl, (C 1 -C 20 ) alkyleno and heteroalkyleno, (C 2 -C 20 ) alkyno and heteroalkyno, or (C 2 -C 20 ) alkeno and heteroalkeno;
R 70 and R 71 , when taken alone, are each independently selected from the group consisting of hydrogen, hydroxyl and a moiety which blocks polymerase-mediated template-directed polymerization, or when taken together form a bond such that the illustrated sugar is 2′,3′-didehydroribose; and
R 72 is selected from the group consisting of hydroxyl, a phosphate ester having the formula
where a is an integer from 0 to 2, and a phosphate ester analog, or a salt thereof.
48 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 where Z has the form Z 1 —L—R 41 —L″—DD/AD—L 3 —R 46 —L′—NUC, or a salt thereof, wherein:
R 41 is a covalent linkage formed upon reaction between a nucleophile and an electrophile;
L″ is a bond or a linker;
DD/AD is a donor dye or an acceptor dye which includes a linking moiety; and.
L 3 is a bond or a linker.
49 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 where Y has the form Y 1 —R 41 —L″—DD/AD—L 3 —R 46 —L′—NUC, or a salt thereof wherein:
Y 1 is Y-1, Y-2, Y-3, or Y-4;
R 41 is a covalent linkage formed upon reaction between a nucleophile and an electrophile;
L″ is a bond or a linker;
DD/AD is a donor dye or an acceptor dye which includes a linking moiety; and.
L 3 is a bond or a linker.
50 . A labeled nucleoside/tide or nucleoside/tide analog of claim 47 where Y has the form Y 1 —R 41 —L″ 13 DD/AD and Z has the form Z 1 —L—R 46 —L′—NUC, or a salt thereof; wherein:
Y 1 is Y-1, Y-2, Y-3, or Y-4;
R 41 is a covalent linkage formed upon reaction between a nucleophile and an electrophile;
L″ is a bond or a linker;
DD/AD is a donor dye or an acceptor dye which includes a linking moiety; and
Z 1 is (C 1 -C 12 ) alkyldiyl, (C 1 -C 12 ) alkyldiyl independently substituted with one or more of the same or different W 1 groups, (C 5 -C 14 ) aryldiyl, and (C 5 -C 14 ) aryldiyl, heteroaryldiyl and heteroaryldiyl independently substituted with one or more of the same or different W 2 groups.
51 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 , 48 , 49 or 50 which is enzymatically incorporatable.
52 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 , 48 , 49 or 50 which is a terminator.
53 . The lableled nucleoside/tide or nucleoside/tide analog of claim 47 , 48 , 49 or 50 which is enzymatically extendable.
54 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 in which L′ is selected from the group consisting of:
propargyl, where the terminal sp carbon is covalently attached to nucleobase B and the terminal methylene (sp 3 ) carbon is covalently attached to F x ; and
—C≡C—CH 2 —O—CH 2 —CH 2 —NR 47 —R 48 —, where R 47 is hydrogen or (C 1 -C 6 ) alkyl and R 48 is —C(O)—(CH 2 ) r —, —C(O)—CHR 49 —, —C(O)—C≡C—CH 2 — or —C(O)-φ-(CH 2 ) r —, where each r is independently an integer from 1 to 5 and φ is C 6 aryldiyl orheteroaryldiyl and R 49 is hydrogen, (C 1 -C 6 ) alkyl or a side chain of an encoding or non-encoding amino acid, and where the terminal sp carbon is covalently attached to nucleobase B and the other terminal group is covalently attached to F x .
55 . The labeled nucleoside/tide or nucleoside/tide analog of claim 48 or claim 49 in which L 3 is a bond, R 46 the formula —C(O)—NHR 51 , where R 51 is hydrogen or (C 1 -C 6 ) alkyl.
56 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 in which nucleobase B is a purine, a 7-deazapurine, an 8-aza, 7-deazapurine, a pyrimidine, a normal nucleobase or a common analog of a normal nucleobase.
57 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 or claim 48 in which Y is selected from the group consisting of Y-1, Y-2, Y-3 and Y-4.
58 . The labeled nucleoside/tide or nucleoside/tide analog of claim 47 or claim 48 in which Y is selected from the group consisting of Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.
59 . The labeled nucleoside/tide or nucleoside/tide analog of claim 49 or claim 50 in which Y 1 is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y4b, Y-1c, Y-2c, Y-3c and Y-4c.
60 . The labeled nucleoside/tide or nucleoside/tide analog of claim 49 or claim 50 in which Y 1 is selected from the group consisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-46b and Y-46c.
61 . A polynucleotide labeled with a rhodamine dye according to claim 1 or an energy-transfer dye pair according to claim 28 .
62 . A method of generating a labeled primer extension product, comprising the step of enzymatically extending a primer-target hybrid in the presence of a mixture of enzymatically-extendable nucleotides capable of supporting continuous primer extension and a terminator, wherein said primer or said terminator is labeled with a rhodamine dye according to claim 1 or an energy-transfer dye pair according to claim 28 .
63 . The method of claim 62 in which the terminator has the structure:
wherein R 70 and R 71 , when taken alone, are each independently selected from the group consisting of hydrogen, halide, and any moiety which blocks polymerase-mediated template-directed polymerization.
64 . The method of claim 62 in which the terminator is a mixture of four different terminators, one which terminates at a template A, one which terminates at a template G, one which terminates at a template C and one which terminates at a template T or U.
65 . The method of claim 62 in which each of the four different terminators is labeled ith a different, spectrally-resolvable fluorophore.
66 . A labelled rhodamine dye-polypeptide conjugate comprising the rhodamine dye of claim 1 and a polypeptide, wherein the polypeptide is selected from the group consisting of a peptide, a protein, and an antibody.
67 . A method of detecting a rhodamine dye-antibody conjugate, in which said conjugate is a rhodamine dye-antibody conjugate according to claim 66 , comprising the steps of:
(a) binding the conjugate to a peptide or protein, and (b) detecting the rhodamine dye-antibody conjugate bound to the peptide or protein.
68 . The method of claim 67 in which the conjugate is bound to the peptide or protein in the presence of a second antibody specific for binding said peptide or protein.
69 . The method of claim 68 in which the second antibody is bound to a solid bead or particle.Join the waitlist — get patent alerts
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