US2002115079A1PendingUtilityA1
Process for detecting target nucleic acid, process for quantifying the same, and pyrylium compound for chemiluminescence analysis
Priority: Oct 3, 1996Filed: Aug 17, 2001Published: Aug 22, 2002
Est. expiryOct 3, 2016(expired)· nominal 20-yr term from priority
C12Q 1/6816C07D 335/02C12Q 1/6813C07D 309/34
56
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
The present invention provides a process for detecting or quantifying a target nucleic acid in a sample, the process comprising the steps of associating a chemiluminescent compound, capable of being associated with a double-stranded nucleic acid, with a double-stranded nucleic acid including the target nucleic acid, and detecting or measuring chemiluminescence derived from the chemiluminescent compound associated with the double-stranded nucleic acid. According to the process, the target nucleic acid in the sample can be highly sensitively detected, or precisely quantified.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A process for detecting a target single-stranded nucleic acid having a first base sequence, said process comprising the steps of:
forming a double-stranded nucleic acid by hybridizing said target single-stranded nucleic acid with a probe nucleic acid having a second base sequence complementary to said first base sequence; providing a chemiluminescent compound capable of being associated with a double-stranded nucleic acid, and then associating said chemiluminescent compound with the double-stranded nucleic acid resulting from said forming step; and detecting luminescence from said chemiluminescent compound associated with said double-stranded nucleic acid.
2 . The process according to claim 1 , wherein the luminescence-detecting step is conducted under a condition that only said chemiluminescent compound associated with said double-stranded nucleic acid can exhibit chemiluminescence.
3 . The process according to claim 2 , wherein said condition is in an aqueous medium in which said chemiluminescent compound non-associated with a double-stranded nucleic acid does not exhibit chemiluminescence.
4 . The process according to claim 3 , wherein said aqueous medium is water.
5 . The process according to claim 3 , wherein said aqueous medium is an aqueous buffer solution.
6 . The process according to claim 3 , wherein said aqueous medium is a mixture solution of water and an organic solvent miscible with water.
7 . The process according to claim 6 , wherein said organic solvent comprises at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, dimethylformamide, dimethylsulfoxide, and isopropanol.
8 . The process according to claim 6 , wherein said mixture solution has a content of said organic solvent falling within 2 to 50% by volume relative to water.
9 . The process according to claim 8 , wherein said content falls within 5 to 20% by volume relative to water.
10 . The process according to claim 3 , wherein pH of said aqueous medium ranges from 5 to 8.
11 . The process according to claim 1 , wherein said chemiluminescent compound is capable of being inserted into the double helical structure of said double-stranded nucleic acid as an intercalator.
12 . The process according to claim 11 , wherein said chemiluminescent compound is a pyrylium compound represented by the following formula [1]:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
13 . The process according to claim 12 , wherein L in said formula [1] is any one of the groups represented by the following formulae [2] to [6], respectively:
wherein Z is a hydrogen atom or a substituted or unsubstituted lower alkyl group, n is 0, 1 or 2, and φ is a substituted or o-, m- or p-phenylene group.
14 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [7]:
wherein X is O, S, Se, or Te, and Y — is an anion.
15 . The process according to claim 14 , wherein X is O or S, and Y is I or ClO 4 .
16 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [8]:
wherein X is O, S, Se, or Te, and Y— is an anion.
17 . The process according to claim 16 , wherein X is O or S, and Y is I or ClO 4 .
18 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [9]:
wherein X is O, S, Se, or Te, and Y— is an anion.
19 . The process according to claim 18 , wherein X is O or S, and Y is I or ClO 4 .
20 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [10]:
wherein X is O, S, Se, or Te, and Y— is an anion.
21 . The process according to claim 20 , wherein X is O or S, and Y is I or ClO 4 .
22 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [11]:
wherein X is O, S, Se, or Te, and Y— is an anion.
23 . The process according to claim 22 , wherein X is O or S, and Y is I or ClO 4 .
24 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [12]:
wherein X is O, S, Se, or Te, and Y— is an anion.
25 . The process according to claim 24 , wherein X is O or S, and Y is I or ClO 4 .
26 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [13]:
wherein X is O, S, Se, or Te, and Y— is an anion.
27 . The process according to claim 26 , wherein X is O or S, and Y is I or ClO 4 .
28 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [14]:
wherein X is O, S, Se, or Te, and Y— is an anion.
29 . The process according to claim 28 , wherein X is O or S, and Y is I or ClO 4 .
30 . The process according to claim 12 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [15]:
wherein X is O, S, Se, or Te, and Y— is an anion.
31 . The process according to claim 30 , wherein X is O or S, and Y is I or ClO 4 .
32 . The process according to claim 12 , wherein at least one hydrophilic group is introduced into at least one substituent of said pyrylium compound.
33 . The process according to claim 1 , wherein said chemiluminescent compound is inserted into said double-stranded nucleic acid by groove binding.
34 . The process according to claim 1 , wherein said luminescence-detecting step includes allowing said chemiluminescent compound and said double-stranded nucleic acid to coexist with an oxalic ester and hydrogen peroxide.
35 . The process according to claim 34 , wherein said oxalic ester is bisdinitrophenyl oxalate.
36 . The process according to claim 1 , wherein said step of forming a double-strand ed nucleic acid includes immobilizing said target nucleic acid or said probe nucleic acid to a solid phase previous to hybridizing said target nucleic acid with said probe nucleic acid.
37 . The process according to claim 1 , wherein said step of forming a double-stranded nucleic acid includes the steps of:
immobilizing said target nucleic acid to a solid phase; preparing as said probe nucleic acid, a single-stranded nucleic acid capable of binding with said target nucleic acid through complementary sequences at 3′-end regions of the target nucleic acid and the single-stranded nucleic acid; hybridizing said target nucleic acid with said probe nucleic acid to form a double-stranded nucleic acid; and polymerizing nucleotides to the 3′-ends of said target nucleic acid and said probe nucleic acid by extension reaction to extend a double-stranded portion in said double-stranded nucleic acid.
38 . The process according to claim 1 , wherein said step of forming a double-stranded nucleic acid includes the steps of:
preparing as said probe nucleic acid, a single-stranded nucleic acid capable of binding with said target nucleic acid through complementary sequences at 3′-end regions of the target nucleic acid and the single-stranded nucleic acid; immobilizing said probe nucleic acid to a solid phase; hybridizing said target nucleic acid with said probe nucleic acid to form a double-stranded nucleic acid; and polymerizing nucleotides to the 3′-ends of said target nucleic acid and said probe nucleic acid by extension reaction to extend a double-stranded portion in said double-stranded nucleic acid.
39 . The process according to claim 36 , 37 or 38 , wherein said solid phase comprises a plastic plate.
40 . The process according to claim 1 , wherein said target nucleic acid is DNA or RNA.
41 . The process according to claim 40 , wherein said DNA is cDNA.
42 . The process according to claim 40 , wherein said RNA is mRNA, tRNA or rRNA.
43 . The process according to claim 1 , wherein said probe nucleic acid is DNA or RNA.
44 . The process according to claim 1 , wherein said target nucleic acid is mRNA comprising a base sequence corresponding to oligoriboadenylic acid at 3′-end region thereof, and said probe nucleic acid comprises a base sequence corresponding to oligodeoxyribothymidylic acid or polydeoxyribothymidylic acid, the base sequence relating to hybridization with said target nucleic acid.
45 . A process for quantifying a target single-stranded nucleic acid having a first base sequence, said process comprising the steps of:
forming a double-stranded nucleic acid by hybridizing said target single-stranded nucleic acid with a probe nucleic acid having a second base sequence complementary to said first base sequence; providing a chemiluminescent compound capable of being associated with a double-stranded nucleic acid, and then associating said chemiluminescent compound with the double-stranded nucleic acid resulting from said forming step; and measuring luminescence from said chemiluminescent compound associated with said double-stranded nucleic acid.
46 . The process according to claim 45 , wherein the luminescence-measuring step is conducted under a condition that only said chemiluminescent compound associated with said double-stranded nucleic acid can exhibit chemiluminescence.
47 . The process according to claim 46 , wherein said condition is in an aqueous medium in which said chemiluminescent compound non-associated with a double-stranded nucleic acid does not exhibit chemiluminescence.
48 . The process according to claim 47 , wherein said aqueous medium is water.
49 . The process according to claim 47 , wherein said aqueous medium is an aqueous buffer solution.
50 . The process according to claim 47 , wherein said aqueous medium is a mixture solution of water and an organic solvent miscible with water.
51 . The process according to claim 50 , wherein said organic solvent comprises at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, dimethylformamide, dimethylsulfoxide, and isopropanol.
52 . The process according to claim 50 , wherein said mixture solution has a content of said organic solvent falling within 2 to 50% by volume relative to water.
53 . The process according to claim 52 , wherein said content falls within 5 to 20% by volume relative to water.
54 . The process according to claim 47 , wherein pH of said aqueous medium ranges from 5 to 8.
55 . The process according to claim 45 , wherein said chemiluminescent compound is capable of being inserted into the double helical structure of said double-stranded nucleic acid as an intercalator.
56 . The process according to claim 55 , wherein said chemiluminescent compound is a pyrylium compound represented by the following formula [1]:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
57 . The process according to claim 56 , wherein L in said formula [1] is any one of the groups represented by the following formulae [2] to [6], respectively:
-φ-(CH═CH)n- [3]; —CH═CH-φ-CH═CH— [4];
wherein Z is a hydrogen atom or a substituted or unsubstituted lower alkyl group, n is 0, 1 or 2, and φ is a substituted or unsubstituted o-, m- or p-phenylene group.
58 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [7]:
wherein X is O, S, Se, or Te, and Y— is an anion.
59 . The process according to claim 58 , wherein X is O or S, and Y is I or ClO 4 .
60 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [8]:
wherein X is O, S, Se, or Te, and Y— is an anion.
61 . The process according to claim 60 , wherein X is O or S, and Y is I or ClO 4 .
62 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [9]:
wherein X is O, S, Se, or Te, and Y— is an anion.
63 . The process according to claim 62 , wherein X is O or S, and Y is I or ClO 4 .
64 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [10]:
wherein X is O, S, Se, or Te, and Y— is an anion.
65 . The process according to claim 64 , wherein X is O or S, and Y is I or ClO 4 .
66 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [11]:
wherein X is O, S, Se, or Te, and Y— is an anion.
67 . The process according to claim 66 , wherein X is O or S, and Y is I or ClO 4 .
68 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [12]:
wherein X is O, S, Se, or Te, and Y— is an anion.
69 . The process according to claim 68 , wherein X is O or S, and Y is I or ClO 4 .
70 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [13]:
wherein X is O, S, Se, or Te, and Y— is an anion.
71 . The process according to claim 70 , wherein X is O or S, and Y is I or ClO 4 .
72 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [14]:
wherein X is O, S, Se, or Te, and Y— is an anion.
73 . The process according to claim 72 , wherein X is O or S, and Y is I or ClO 4 .
74 . The process according to claim 56 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [15]:
wherein X is O, S, Se, or Te, and Y— is an anion.
75 . The process according to claim 74 , wherein X is O or S, and Y is I or ClO 4 .
76 . The process according to claim 56 , wherein at least one hydrophilic group is introduced into at least one substituent of said pyrylium compound.
77 . The process according to claim 45 , wherein said chemiluminescent compound is inserted into said double-stranded nucleic acid by groove binding.
78 . The process according to claim 45 , wherein said luminescence-measuring step includes allowing said chemiluminescent compound and said double-stranded nucleic acid to coexist with an oxalic ester and hydrogen peroxide.
79 . The process according to claim 78 , wherein said oxalic ester is bisdinitrophenyl oxalate.
80 . The process according to claim 45 , wherein said step of forming a double-stranded nucleic acid includes immobilizing said target nucleic acid or said probe nucleic acid to a solid phase previous to hybridizing said target nucleic acid with said probe nucleic acid.
81 . The process according to claim 45 , wherein said step of forming a double-stranded nucleic acid includes the steps of:
immobilizing said target nucleic acid to a solid phase; preparing as said probe nucleic acid, a single-stranded nucleic acid capable of binding with said target nucleic acid through complementary sequences at 3′-end regions of the target nucleic acid and the single-stranded nucleic acid; hybridizing said target nucleic acid with said probe nucleic acid to form a double-stranded nucleic acid; and polymerizing nucleotides to the 3′-ends of said target nucleic acid and said probe nucleic acid by extension reaction to extend a double-stranded portion in said double-stranded nucleic acid.
82 . The process according to claim 45 , wherein said step of forming a double-stranded nucleic acid includes the steps of:
preparing as said probe nucleic acid, a single-stranded nucleic acid capable of binding with said target nucleic acid through complementary sequences at 3′-end regions of the target nucleic acid and the single-stranded nucleic acid; immobilizing said probe nucleic acid to a solid phase; hybridizing said target nucleic acid with said probe nucleic acid to form a double-stranded nucleic acid; and polymerizing nucleotides to the 3′-ends of said target nucleic acid and said probe nucleic acid by extension reaction to extend a double-stranded portion in said double-stranded nucleic acid.
83 . The process according to claim 80 , 81 or 82 , wherein said solid phase comprises a plastic plate.
84 . The process according to claim 45 , wherein said target nucleic acid is DNA or RNA.
85 . The process according to claim 84 , wherein said DNA is cDNA.
86 . The process according to claim 84 , wherein said RNA is mRNA, tRNA or rRNA.
87 . The process according to claim 45 , wherein said probe nucleic acid is DNA or RNA.
88 . The process according to claim 45 , wherein said target nucleic acid is MRNA comprising a base sequence corresponding to oligoriboadenylic acid at 3′-end region thereof, and said probe nucleic acid comprises a base sequence corresponding to oligodeoxyribothymidylic acid or polydeoxyribothymidylic acid, the base sequence relating to hybridization with said target nucleic acid.
89 . A process for detecting a target double-stranded nucleic acid comprising the steps of:
providing a chemiluminescent compound capable of being associated with a double-stranded nucleic acid, and then associating said chemiluminescent compound with the target double-stranded nucleic acid; and detecting luminescence from said chemiluminescent compound associated with said target double-stranded nucleic acid.
90 . The process according to claim 89 , wherein the luminescence-detecting step is conducted under a condition that only said chemiluminescent compound associated with said double-stranded nucleic acid can exhibit chemiluminescence.
91 . The process according to claim 90 , wherein said condition is in an aqueous medium in which said chemiluminescent compound non-associated with a double-stranded nucleic acid does not exhibit chemiluminescence.
92 . The process according to claim 91 , wherein said aqueous medium is water.
93 . The process according to claim 91 , wherein said aqueous medium is an aqueous buffer solution.
94 . The process according to claim 91 , wherein said aqueous medium is a mixture solution of water and an organic solvent miscible with water.
95 . The process according to claim 94 , wherein said organic solvent comprises at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, dimethylformamide, dimethylsulfoxide, and isopropanol.
96 . The process according to claim 94 , wherein said mixture solution has a content of said organic solvent falling within 2 to 50% by volume relative to water.
97 . The process according to claim 96 , wherein said content falls within 5 to 20% by volume relative to water.
98 . The process according to claim 91 , wherein pH of said aqueous medium ranges from 5 to 8.
99 . The process according to claim 89 , wherein said chemiluminescent compound is capable of being inserted into the double helical structure of said double-stranded nucleic acid as an intercalator.
100 . The process according to claim 99 , wherein said chemiluminescent compound is a pyrylium compound represented by the following formula [1]:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
101 . The process according to claim 100 , wherein L in said formula [1] is any one of the groups represented by the following formulae [2] to [6], respectively:
-φ-(CH═CH) n- [3]; —CH═CH-φ-CH═CH— [];
wherein Z is a hydrogen atom or a substituted or unsubstituted lower alkyl group, n is 0, 1 or 2, and φ is a substituted or unsubstituted o-, m- or p-phenylene group.
102 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [7]:
wherein X is O, S, Se, or Te, and Y— is an anion.
103 . The process according to claim 102 , wherein X is or S, and Y is I or ClO 4 .
104 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is compound represented by the following formula [8]:
wherein X is O, S, Se, or Te, and Y— is an anion.
105 . The process according to claim 104 , wherein X is or S, and Y is I or ClO 4 .
106 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [9]:
wherein X is O, S, Se, or Te, and Y— is an anion.
107 . The process according to claim 106 , wherein X is or S, and Y is I or ClO 4 .
108 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [10]:
wherein X is O, S, Se, or Te, and Y— is an anion.
109 . The process according to claim 108 , wherein X is or S, and Y is I or ClO 4 .
110 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [11]:
wherein X is O, S, Se, or Te, and Y— is an anion.
111 . The process according to claim 110 , wherein X is or S, and Y is I or ClO 4 .
112 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [12]:
wherein X is O, S, Se, or Te, and Y— is an anion.
113 . The process according to claim 112 , wherein X is O or S, and Y is I or ClO 4 .
114 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [13]:
wherein X is O, S, Se, or Te, and Y— is an anion.
115 . The process according to claim 114 , wherein X is O or S, and Y is I or ClO 4 .
116 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [14]:
wherein X is O, S, Se, or Te, and Y— is an anion.
117 . The process according to claim 116 , wherein X is O or S, and Y is I or ClO 4 .
118 . The process according to claim 100 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [15]:
wherein X is O, S, Se, or Te, and Y— is an anion.
119 . The process according to claim 118 , wherein X is O or S, and Y is I or ClO 4 .
120 . The process according to claim 100 , wherein at least one hydrophilic group is introduced into at least one substituent of said pyrylium compound.
121 . The process according to claim 89 , wherein said luminescence-detecting step includes allowing said chemiluminescent compound and said double-stranded nucleic acid to coexist with an oxalic ester and hydrogen peroxide.
122 . The process according to claim 121 , wherein said oxalic ester is bisdinitrophenyl oxalate.
123 . The process according to claim 89 , wherein said chemiluminescent compound is inserted into said double-stranded nucleic acid by groove binding.
124 . A process for quantifying a target double-stranded nucleic acid comprising the steps of:
providing a chemiluminescent compound capable of being associated with a double-stranded nucleic acid, and then associating said chemiluminescent compound with the target double-stranded nucleic acid; and measuring luminescence from said chemiluminescent compound associated with said target double-stranded nucleic acid.
125 . The process according to claim 124 , wherein the luminescence-measuring step is conducted under a condition that only said chemiluminescent compound associated with said double-stranded nucleic acid can exhibit chemiluminescence.
126 . The process according to claim 125 , wherein said condition is in an aqueous medium in which said chemiluminescent compound non-associated with a double-stranded nucleic acid does not exhibit chemiluminescence.
127 . The process according to claim 126 , wherein said aqueous medium is water.
128 . The process according to claim 126 , wherein said aqueous medium is an aqueous buffer solution.
129 . The process according to claim 126 , wherein said aqueous medium is a mixture solution of water and an organic solvent miscible with water.
130 . The process according to claim 129 , wherein said organic solvent comprises at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, dimethylformamide, dimethylsulfoxide, and isopropanol.
131 . The process according to claim 129 , wherein said mixture solution has a content of said organic solvent falling within 2 to 50% by volume relative to water.
132 . The process according to claim 131 , wherein said content falls within 5 to 20% by volume relative to water.
133 . The process according to claim 126 , wherein pH of said aqueous medium ranges from 5 to 8.
134 . The process according to claim 124 , wherein said chemiluminescent compound is capable of being inserted into the double helical structure of said double-stranded nucleic acid as an intercalator.
135 . The process according to claim 134 , wherein said chemiluminescent compound is a pyrylium compound represented by the following formula [1]:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
136 . The process according to claim 135 , wherein L in said formula [1] is any one of the groups represented by the following formulae [2] to [6], respectively:
-φ- (CH═CH) n- [3]; —CH═CH-φ-CH═CH— [4];
wherein Z is a hydrogen atom or a substituted or unsubstituted lower alkyl group, n is 0, 1 or 2, and φ is a substituted or unsubstituted o-, m- or p-phenylene group.
137 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [7]:
wherein X is O, S, Se, or Te, and Y— is an anion.
138 . The process according to claim 137 , wherein X is O or S, and Y is I or ClO 4 .
139 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [8]:
wherein X is O, S, Se, or Te, and Y— is an anion.
140 . The process according to claim 139 , wherein X is O or S, and Y is I or ClO 4 .
141 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [9]:
wherein X is O, S, Se, or Te, and Y— is an anion.
142 . The process according to claim 141 , wherein X is or S, and Y is I or ClO 4 .
143 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is a compound represented by the following formula [10]:
wherein X is O, S, Se, or Te, and Y— is an anion.
144 . The process according to claim 143 , wherein X is O or S, and Y is I or ClO 4 .
145 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [11]:
wherein X is O, S, Se, or Te, and Y— is an anion.
146 . The process according to claim 145 , wherein X is O or S, and Y is I or ClO 4 .
147 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [12]:
wherein X is O, S, Se, or Te, and Y— is an anion.
148 . The process according to claim 147 , wherein X is O or S, and Y is I or ClO 4 .
149 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [13]:
wherein X is O, S, Se, or Te, and Y— is an anion.
150 . The process according to claim 149 , wherein X is O or S, and Y is I or ClO 4 .
151 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [14]:
wherein X is O, S, Se, or Te, and Y— is an anion.
152 . The process according to claim 151 , wherein X is or S, and Y is I or ClO 4 .
153 . The process according to claim 135 , wherein said chemiluminescent compound represented by said formula [1] is the compound represented by the following formula [15]:
wherein X is O, S, Se, or Te, and Y— is an anion.
154 . The process according to claim 153 , wherein X is O or S, and Y is I or ClO 4 .
155 . The process according to claim 135 , wherein at least one hydrophilic group is introduced into at least one substituent of said pyrylium compound.
156 . The process according to claim 124 , wherein said luminescence-measuring step includes allowing said chemiluminescent compound and said double-stranded nucleic acid to coexist with an oxalic ester and hydrogen peroxide.
157 . The process according to claim 156 , wherein said oxalic ester is bisdinitrophenyl oxalate.
158 . The process according to claim 124 , wherein said chemiluminescent compound is inserted into said double-stranded nucleic acid by groove binding.
159 . A process for detecting a target single-stranded nucleic acid having a first base sequence, said process comprising the steps of:
forming a double-stranded nucleic acid by hybridizing said target single-stranded nucleic acid with a probe nucleic acid having a second base sequence complementary to said first base sequence; providing a compound which can be intercalated into a double-stranded nucleic acid and which is capable of exhibiting chemiluminescence only in a hydrophobic condition, and then intercalating said compound into the double-stranded nucleic acid resulting from said forming step; and placing in an aqueous medium said double-stranded nucleic acid into which said compound is intercalated together with a reagent capable of causing said compound to exhibit chemiluminescence, and detecting the resulting chemiluminescence.
160 . A process for quantifying a target single-stranded nucleic acid having a first base sequence, said process comprising the steps of:
forming a double-stranded nucleic acid by hybridizing said target single-stranded nucleic acid with a probe nucleic acid having a second base sequence complementary to said first base sequence; providing a compound which can be intercalated into a double-stranded nucleic acid and which is capable of exhibiting chemiluminescence only in a hydrophobic condition, and then intercalating said compound into the double-stranded nucleic acid resulting from said forming step; and placing in an aqueous medium said double-stranded nucleic acid into which said compound is intercalated together with a reagent capable of causing said compound to exhibit chemiluminescence, and measuring the resulting chemiluminescence.
161 . A process for detecting a target double-stranded nucleic acid comprising the steps of:
providing a compound which can be intercalated into a double-stranded nucleic acid and which is capable of exhibiting substantial chemiluminescence only in a hydrophobic condition, and then intercalating said compound into said target double-stranded nucleic acid; and placing in an aqueous medium said double-stranded nucleic acid into which said compound is intercalated together with a reagent capable of causing said compound to exhibit chemiluminescence, and detecting the resulting chemiluminescence.
162 . A process for quantifying a target double-stranded nucleic acid comprising the steps of:
providing a compound which can be intercalated into a double-stranded nucleic acid and which is capable of exhibiting substantial chemiluminescence only in a hydrophobic condition, and then intercalating said compound into said target double-stranded nucleic acid; and placing in an aqueous medium said double-stranded nucleic acid into which said compound is intercalated together with a reagent capable of causing said compound to exhibit chemiluminescence, and measuring the resulting chemiluminescence.
163 . The detecting process according to claim 159 or 161 , wherein said compound is a pyrylium compound represented by the following formula [1]:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
164 . The quantifying process according to claim 160 or 162 , wherein said compound is a pyrylium compound represented by the following formula [1]:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
165 . A pyrylium compound represented by the following formula [1] for use in chemiluminescence analysis:
wherein:
X is O, S, Se or Te;
two of R 1 , R 2 and R 3 are independently a substituted or unsubstituted aryl group;
the other is a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted alkyl group, substituted or unsubstituted cycloalkyl group, -A or -L-A, wherein:
L is -L 1 -, -L 2 -L 3 - or -L 4 -L 5 -L 6 -, wherein each of L 1 to L 6 is independently —(CH═CH)—, a divalent group derived from the substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or —CH═R 4 —, wherein R 4 is a ring structure having an oxo group; and
A is a substituted or unsubstituted aryl group, or —CH═R 5 , wherein R 5 is a substituted or unsubstituted heterocyclic ring, substituted or unsubstituted cycloalkyl group or substituted or unsubstituted aromatic ring; and
Y— is an anion.
166 . The pyrylium compound according to claim 165 , wherein L in said formula [1] is any one of the groups represented by the following formulae [2] to [6], respectively:
-φ-(CH═CH) n- [3]; —CH═CH-φ-CH═CH— [4];
wherein Z is a hydrogen atom or a substituted or unsubstituted lower alkyl group, n is 0, 1 or 2, and φ is a substituted or unsubstituted o-, m- or p-phenylene group.
167 . The pyrylium compound according to claim 165 , represented by the following formula [7]:
wherein X is O, S, Se, or Te, and Y— is an anion.
168 . The pyrylium compound according to claim 167 , wherein X is O or S, and Y is I or ClO 4 .
169 . The pyrylium compound according to claim 165 , represented by the following formula [8]:
wherein X is O, S, Se, or Te, and Y— is an anion.
170 . The pyrylium compound according to claim 169 , wherein X is O or S, and Y is I or ClO 4 .
171 . The pyrylium compound according to claim 165 , represented by the following formula [9]:
wherein X is O, S, Se, or Te, and Y— is an anion.
172 . The pyrylium compound according to claim 171 , wherein X is O or S, and Y is I or ClO 4 .
173 . The pyrylium compound according to claim 165 , represented by the following formula [10]:
wherein X is O, S, Se, or Te, and Y— is an anion.
174 . The pyrylium compound according to claim 173 , wherein X is O or S, and Y is I or ClO 4 .
175 . The pyrylium compound according to claim 165 , represented by the following formula [11]:
wherein X is O, S, Se, or Te, and Y— is an anion.
176 . The pyrylium compound according to claim 175 , wherein X is O or S, and Y is I or ClO 4 .
177 . The pyrylium compound according to claim 165 , represented by the following formula [12]:
wherein X is O, S, Se, or Te, and Y— is an anion.
178 . The pyrylium compound according to claim 177 , wherein X is O or S, and Y is I or ClO 4 .
179 . The pyrylium compound according to claim 165 , represented by the following formula [13]:
wherein X is O, S, Se, or Te, and Y— is an anion.
180 . The pyrylium compound according to claim 179 , wherein X is O or S, and Y is I or ClO 4 .
181 . The pyrylium compound according to claim 165 , represented by the following formula [14]:
wherein X is O, S, Se, or Te, and Y— is an anion.
182 . The pyrylium compound according to claim 181 , wherein X is O or S, and Y is I or ClO 4 .
183 . The pyrylium compound according to claim 165 , represented by the formula [15]:
wherein X is O, S, Se, or Te, and Y— is an anion.
184 . The pyrylium compound according to claim 183 , wherein X is O or S, and Y is I or ClO 4 .
185 . The pyrylium compound according to claim 165 , wherein at least one hydrophilic group is introduced into at least one substituent of said pyrylium compound.Join the waitlist — get patent alerts
Track US2002115079A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.