Novel dyes for the detection or quantification of desirable target molecules

• US 20060269926A1
• Filed: 05/24/2005
• Published: 11/30/2006
• Est. Priority Date: 05/24/2005
• Status: Active Grant

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Dakai Liu, Elazar Rabbani, Praveen Pande, Rajesh Khazanchi, Wei Cheng, Yuejun Xiang
• Inventors: Rabbani, Elazar, Cheng, Wei, Pande, Praveen, Liu, Dakai, Xiang, Yuejun, Khazanchi, Rajesh
• Granted Patent: US 7,569,695 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 235/20 Two benzimidazolyl-2 radicals linked together directly or via a hydrocarbon or substituted hydrocarbon radical
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/14 containing three or more hetero rings
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C09B 23/0091 having only one heterocyclic ring at one end of the methine chain, e.g. hemicyamines, hemioxonol styryl dyes see C09B23/14
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/105 two >CH- groups
• CPC Class Codes: C09B 23/145 the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
• CPC Class Codes: C09B 23/146 (Benzo)thiazolstyrylamino dyes

Abstract

The present invention provides dyes, reactive dyes and labeled reagents that may be used in the detection or quantification of desirable target molecules, such as proteins and nucleic acids. Dyes are provided that may be used free in solution where the binding of the dye to the target molecule provides signal generation. Dyes are also provided that comprise reactive groups that may be used to attach the dyes to probes that will bind to desirable target molecules. The novel dyes of the present invention have been modified by the addition of charged and polar groups to provide beneficial properties.

Claims

1-102. -102. (canceled)

103. A dye having the formula:

104. The dye of claim 103 wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹³, R¹⁴, or R¹⁵ comprises a reactive group.

105. The dye of claim 104 wherein said reactive group comprises a nucleophilic reactive group, an electrophilic reactive group, a terminal alkene, a terminal alkyne, a coordinate group or an alkylating agent.

106. The dye of claim 105 wherein said nucleophilic reactive group comprises a thiol, amine or hydroxyl group.

107. The dye of claim 105 wherein said electrophilic reactive group comprises an isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde group.

108. The dye of claim 103, having the following structure:

109. The dye of claim 108, having the following structure:

110. The dye of claim 108, having the following structure:

111. The dye of claim 110, having the following structure:

112. The dye of claim 110, having the following structure:

113. The dye of claim 112, having the following structure:

114. The dye of claim 103, having the following structure:

115. The dye of claim 103, wherein said dye further comprises one or more charged groups or polar groups.

116. The dye of claim 115, wherein at least one of said one or more charged groups or polar groups is monomeric.

117. The dye of claim 115, wherein at least one of said one or more charged groups or polar groups is polymeric.

118. The dye of claim 115 wherein at least one of said one or more charged groups or polar groups is anionic.

119. The dye of claim 115, wherein at least one of said one or more charged groups or polar groups is cationic.

120. The dye of claim 103, wherein said dye further comprises one or more anionic charged groups or polar groups and one or more cationic charged groups or polar groups.

121. The dye of claim 108, having the following structure

122. The dye of claim 110, having the following structure:

123. The dye of claim 112, having the following structure:

124. The dye of claim 114, having the following structure:

125. The dye of claim 103, wherein said dye is linked to a target molecule.

126. The dye of claim 125, wherein said target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, plastic particle, plastic surface, silicaceous particle, silicaceous surface, organic molecule, dye or a derivative thereof.

127. The dye of claim 126, wherein said nucleoside, nucleotide, oligonucleotide, or polynucleotide comprises one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues or any combination thereof.

128. The dye of claim 126, wherein said dye is linked to said target molecule through a linker arm.

129. The dye of claim 128, wherein said linker arm is attached to said target molecule through a bond which comprises a covalent bond, a non-covalent bond, a polar bond or a coordinate bond.

130. The oligonucleotide or polynucleotide of claim 126, wherein said oligonucleotide or polynucleotide comprises two or more dye molecules attached to separate nucleotides of said oligonucleotide or polynucleotide.

131. The oligonucleotide or polynucleotide of claim 130, wherein said two or more dye molecules comprise the same dye molecules.

132. The oligonucleotide or polynucleotide of claim 130, wherein said two or more dye molecules comprise different dye molecules.

133. A composite dye comprising the dye of claim 126, said dye being joined or attached to a second dye molecule.

134. The dye of claim 103, wherein said dye is attached to a target specific moiety.

135. The dye of claim 134, wherein said target specific moiety comprises a protein or a nucleic acid.

136. The dye of claim 135, wherein said protein comprises an antibody or a fragment thereof.

137. The dye of claim 135, wherein said nucleic acid comprises unmodified nucleotides, modified nucleotides, nucleotide analogues or any combination thereof.

138. The dye of claim 111, having the following structure:

140. The dye of claim 111, having the following structure:

142. The dye of claim 103, wherein said alkyl or alkoxy groups independently comprise from 1-18 carbons.

143. The dye of claim 142, wherein said alkyl or alkoxy groups independently comprise from 1-6 carbons.

164. The oligonucleotide or polynucleotide of claim 130, wherein said two or more dye molecules comprise different dye molecules.

144. A dye having the formula:

145. The dye of claim 144, wherein said nucleophilic reactive group comprises a thiol, amine or hydroxyl group.

146. The dye of claim 144, wherein said electrophilic reactive group comprises an isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide-ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde group.

147. The dye of claim 144, wherein said dye further comprises one or more charged groups or polar groups.

148. The dye of claim 147, wherein at least one of said one or more charged groups or polar groups is monomeric.

149. The dye of claim 147, wherein at least one of said one or more charged groups or polar groups is polymeric.

150. The dye of claim 147, wherein at least one of said one or more charged groups or polar groups is anionic.

151. The dye of claim 147, wherein at least one of said one or more charged groups or polar groups is cationic.

152. The dye of claim 144, wherein said dye further comprises one or more anionic charged groups or polar groups and one or more cationic charged groups or polar groups.

153. The dye of claim 144, having the following structure:

154. The dye of claim 144, having the following structure:

155. The dye of claim 144 having the following structure:

156. The dye of claim 144 having the following structure:

157. The dye of claim 144, wherein said dye has been linked to a target molecule through said reactive group.

158. The dye of claim 144, wherein said target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, plastic particle, plastic surface, silicaceous particle, silicaceous surface, organic molecule, dye or a derivative thereof.

159. The dye of claim 158, wherein said nucleoside, nucleotide, oligonucleotide, or polynucleotide comprises one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues or any combination thereof.

160. The dye of claim 157, wherein said dye is linked to said target molecule through a linker arm.

161. The dye of claim 160, wherein said linker arm is attached to said target molecule through a bond which comprises a covalent bond, a non-covalent bond, a polar bond or a coordinate bond.

162. The oligonucleotide or polynucleotide of claim 158, wherein said oligonucleotide or polynucleotide comprises two or more dye molecules attached to separate nucleotides of said oligonucleotide or polynucleotide.

163. The oligonucleotide or polynucleotide of claim 162, wherein said two or more dye molecules comprise the same dye molecules.

165. A composite dye comprising the dye of claim 158, wherein said target molecule comprises a dye.

166. The composite dye of claim 165 further comprising a nucleophilic reactive group, an electrophilic reactive group, a terminal alkene, a terminal alkyne, a coordinate group or an alkylating agent.

167. The dye of claim 144, wherein said dye has been linked to a target specific moiety through said reactive group.

168. The dye of claim 167, wherein said target specific moiety comprises a protein or a nucleic acid.

169. The dye of claim 168, wherein said protein comprises an antibody or a fragment thereof.

170. The dye of claim 168, wherein said nucleic acid comprises unmodified nucleotides, modified nucleotides, nucleotide analogues or any combination thereof.

171. The dye of claim 144, wherein said alkyl or alkoxy groups independently comprise from 1-18 carbons.

172. The dye of claim 171, wherein said alkyl or alkoxy groups independently comprise from 1-6 carbons.

189. The composition of claim 156, wherein said second portion comprises a target specific moiety.

190. The composition of claim 189, wherein said target specific moiety comprises a protein or a nucleic acid.

191. The composition of claim 190, wherein said protein comprises an antibody or a fragment thereof.

192. The composition of claim 190, wherein said nucleic acid comprises unmodified nucleotides, modified nucleotides, nucleotide analogues or any combination thereof.

173. A composition comprising a first portion and a second portion wherein said first portion comprises a dye and said second portion comprises a target molecule, said dye having the formula:

174. The dye of claim 173, wherein said dye further comprises one or more charged groups or polar groups.

175. The dye of claim 174, wherein at least one of said one or more charged groups or polar groups is monomeric.

176. The dye of claim 174, wherein at least one of said one or more charged groups or polar groups is polymeric.

177. The dye of claim 174, wherein at least one of said one or more charged groups or polar groups is anionic.

178. The dye of claim 174, wherein at least one of said one or more charged groups or polar groups is cationic.

179. The dye of claim 173, wherein said dye further comprises one or more anionic charged groups or polar groups and one or more cationic charged groups or polar groups.

180. The dye of claim 173, wherein said target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, plastic particle, plastic surface, silicaceous particle, silicaceous surface, organic molecule, dye or a derivative thereof.

181. The dye of claim 180, wherein said nucleoside, nucleotide, oligonucleotide, or polynucleotide comprises one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues or any combination thereof.

184. The oligonucleotide or polynucleotide of claim 180, wherein said oligonucleotide or polynucleotide comprises two or more dye molecules attached to separate nucleotides of said oligonucleotide or polynucleotide.

185. The oligonucleotide or polynucleotide of claim 184, wherein said two or more dye molecules comprise the same dye molecules.

186. The oligonucleotide or polynucleotide of claim 184, wherein said two or more dye molecules comprise different dye molecules.

187. A composite dye comprising the composition of claim 180, wherein said target molecule comprises a dye.

188. The composite dye of claim 187 further comprising a nucleophilic reactive group, an electrophilic reactive group, a terminal alkene, a terminal alkyne, a coordinate group or an alkylating agent.

193. The composition of claim 173, wherein said alkyl or alkoxy groups independently comprise from 1-18 carbons.

194. The composition of claim 193, wherein said alkyl or alkoxy groups independently comprise from 1-6 carbons.

183. The dye of claim 173, wherein said dye is linked to said target molecule through a linker arm.

183-1. The dye of claim 182, wherein said linker arm is attached to said target molecule through a bond which comprises a covalent bond, a non-covalent bond, a polar bond or a coordinate bond.

195. A method for detecting the presence or quantity of a target comprising the steps of:

a) providing

i) a sample where the presence or quantity of a target is desired to be detected

ii) a composition comprising a first portion and a second portion wherein said first portion comprises a dye and said second portion comprises a target specific moiety, said dye having the formula;

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹² independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R¹¹ and R¹² form a five- or six-membered ring;

wherein n can be 0, 1, 2 or 3;

wherein Y is —

CR⁹═

CR¹⁰—

;

wherein m and p can have values of 0 or 1 and m+p=1;

wherein at least one of R¹, R², R³, R⁴, R⁵, R¹¹ or R¹² comprises Q(1) or at least one of R⁶, R⁷, R⁸, R⁹ or R¹⁰ comprises Q(2);

wherein Q(1) comprises a sulfonate (SO₃⁻

), a sulfonate ester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄⁼), a phosphate monoester (PO₃⁻

ER¹³), a phosphate diester (PO₂ER³ER⁴), a phosphonate (PO₃⁼) a phosphonate monoester (PO₂⁻

ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂⁻

ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻

ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴, NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independently comprise O or S;

wherein Q(1) is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted, or any combinations thereof and wherein when Q(1) is a sulfonamide, it does not comprise a terminal reactive group or a linker joining the dye to a target molecule;

wherein Q(2) comprises a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphonate (PO₃⁼), a phosphonate monoester (PO₂⁻

ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂⁻

ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻

ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independently comprise O or S;

wherein Q(2) is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof and wherein when Q(2) is a sulfonamide, it does not comprise a terminal reactive group or a linker joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can be hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R¹³ and R¹⁴ form a five- or six-membered ring;

wherein R¹⁶, R¹⁷, R¹⁸ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² can independently be hydrogen, Z, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group that is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R² and R³, R³ and R⁴, R⁴ and R⁵, R⁶ and R⁷, and R⁷ and R⁸ may form a five- or six-membered ring;

or when taken together R¹ and R¹⁶, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁷ and R¹⁸, and R¹⁸ and R⁹ may form a five- or six-membered ring;

wherein Z comprises a carboxyl group (CO₂⁻

), a carbonate ester (COER¹³), a sulfonate (SO₃⁻

), a sulfonate ester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄⁼), a phosphate monoester (PO₃⁻

ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃⁼) a phosphonate monoester (PO₂⁻

ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂⁻

ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻

ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴) where E can be independently O or S and the structures are as described previously;

wherein Z is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof;

wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰, R¹¹ or R¹² may further comprise a heteroatom containing side chain wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (—

OR²⁵), a thioether linkage (—

SR²⁵), or an amine linkage (—

NR²⁵R²⁶ or —

N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independently comprise hydrogen, Z, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group that is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five- or six-membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may further comprise said heteroatom containing side chain;

and wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹³, R¹⁴, or R¹⁵ is linked to said second portion.

b) allowing any targets present in said sample i) to bind with said target specific moiety comprising said composition ii); and

c) quantifying the amount of said composition ii) bound to any of said target in the sample, thereby detecting the presence or quantity of said target.

196. The method of claim 195, wherein said target comprises an oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, organic molecule, or a derivative thereof.

197. The method of claim 195, wherein said dye further comprises one or more charged groups or polar groups.

198. The method of claim 197, wherein at least one of said one or more charged groups or polar groups is monomeric.

199. The method of claim 197, wherein at least one of said one or more charged groups or polar groups is polymeric.

200. The method of claim 197, wherein at least one of said one or more charged groups or polar groups is anionic.

201. The method of claim 197, wherein at least one of said one or more charged groups or polar groups is cationic.

202. The method of claim 195, wherein said dye further comprises one or more anionic charged groups or polar groups and one or more cationic charged groups or polar groups.

203. The method of claim 195, wherein said target specific moiety comprises an oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, natural drug, synthetic drug, organic molecule, or a derivative thereof.

204. The method of claim 195, wherein said dye is linked to said target specific moiety through a linker arm.

205. The method of claim 204, wherein said linker arm is attached to said target specific moiety through a bond which comprises a covalent bond, a polar bond or a coordinate bond.

206. The method of claim 203, wherein said oligonucleotide or polynucleotide comprises two or more dye molecules attached to separate nucleotides of said oligonucleotide or polynucleotide.

207. The method of claim 206, wherein said two or more dye molecules comprise the same dye molecules.

208. The method of claim 206, wherein said two or more dye molecules comprise different dye molecules.

209. The method of claim 195, wherein said dye is part of a composite dye comprising a second dye molecule.

210. The method of claim 203, wherein said oligonucleotide or polynucleotide comprises unmodified nucleotides, modified nucleotides, nucleotide analogues or any combination thereof.

211. The method of claim 195, wherein said alkyl or alkoxy groups independently comprise from 1-18 carbons.

212. The method of claim 211, wherein said alkyl or alkoxy groups independently comprise from 1-6 carbons.

213. The method of claim 195, wherein said sample i) or said composition ii) is fixed or immobilized to a solid support.

214. The method of claim 213, further comprising carrying out one or more washing steps after said binding step b).

215. The method of claim 195, wherein said binding step b) and said detecting step c) are carried out in the absence of washing steps.

216. The method of claim 215, wherein said detection step c) is carried out by energy transfer.

217. A method for detecting the presence, location or quantity of a target comprising the steps of:

a) providing

i) a sample where the presence, location or quantity of a target is desired to be detected

ii) a dye, having the formula;

wherein X comprises CR¹¹R¹², NR¹¹, O, S or Se where R¹¹ and R¹² independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R¹¹ and R¹² form a five- or six-membered ring;

wherein n can be 0, 1, 2 or 3;

wherein Y is —

CR⁹═

CR¹⁰—

;

wherein m and p can have values of 0 or 1 and m+p=1;

wherein at least one of R¹, R², R³, R⁴, R⁵, R¹¹ or R¹² comprises Q(1) or at least one of R⁶, R⁷, R⁸, R⁹ or R¹⁰ comprises Q(2);

wherein Q(1) comprises a sulfonate (SO₃⁻

), a sulfonate ester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄⁼), a phosphate monoester (PO₃⁻

ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃⁼) a phosphonate monoester (PO₂⁻

ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂⁻

ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻

ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independently comprise O or S;

wherein Q(1) is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted, or any combinations thereof and wherein when Q(1) is a sulfonamide, it does not comprise a terminal reactive group or a linker joining the dye to a target molecule;

wherein Q(2) comprises a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphonate (PO₃⁼), a phosphonate monoester (PO₂⁻

ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂⁻

ER¹³) a thiophosphate diester (PSOER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻

ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴) wherein any of E can independently comprise O or S;

wherein Q(2) is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof and wherein when Q(2) is a sulfonamide, it does not comprise a terminal reactive group or a linker joining the dye to a target molecule;

wherein R¹³, R¹⁴, R¹⁵, R¹⁹ and R²⁰ can be hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R¹³ and R¹⁴ form a five- or six-membered ring;

wherein R¹⁶, R¹⁷, R¹⁸ and the remaining R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² can independently be hydrogen, Z, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group that is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R² and R³, R³ and R⁴, R⁴ and R⁵, R⁶ and R⁷, and R⁷ and R⁸ may form a five- or six-membered ring;

or when taken together R¹ and R¹⁶, R¹¹ and R¹⁶, R¹⁶ and R¹⁷, R¹⁷ and R¹⁸, and R¹⁸ and R⁹ may form a five- or six-membered ring;

wherein Z comprises a carboxyl group (CO₂⁻

), a carbonate ester (COER¹³), a sulfonate (SO₃⁻

), a sulfonate ester (SO₂ER¹³), a sulfoxide (SOR¹³), a sulfone (SO₂CR¹³R¹⁴R¹⁵), a sulfonamide (SO₂NR¹³R¹⁴), a phosphate (PO₄⁼), a phosphate monoester (PO₃⁻

ER¹³), a phosphate diester (PO₂ER¹³ER¹⁴), a phosphonate (PO₃⁼) a phosphonate monoester (PO₂⁻

ER¹³) a phosphonate diester (POER¹³ER¹⁴), a thiophosphate (PSO₃⁼), a thiophosphate monoester (PSO₂⁻

ER¹³) a thiophosphate diester (PSO⁻

ER¹³ER¹⁴), a thiophosphonate (PSO₂⁼), a thiophosphonate monoester (PSO⁻

ER¹³) a thiophosphonate diester (PSER¹³ER¹⁴), a phosphonamide (PONR¹³R¹⁴NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁹R²⁰), a phosphoramide (PONR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), its thioanalogue (PSNR¹³R¹⁴NR¹⁵NR¹⁹R²⁰), a phosphoramidite (PO₂R¹⁹NR¹³R¹⁴) or its thioanalogue (POSR¹⁹NR¹³R¹⁴) where E can be independently O or S;

wherein Z is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, or any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted or any combinations thereof;

and wherein any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰, R¹¹ or R¹² may further comprise a heteroatom containing side chain wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (—

OR²⁵), a thioether linkage (—

SR²⁵), or an amine linkage (—

NR²⁵R²⁶ or —

N⁺R²⁵R²⁶R²⁷), and wherein R²⁵, R²⁶ and R²⁷ independently comprise hydrogen, Z, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group that is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together, R²⁵ and R²⁶, and R²⁶ and R²⁷ independently comprise a five- or six-membered ring, and wherein any of R²⁵, R²⁶ or R²⁷ may further comprise said heteroatom containing side chain;

b) allowing any targets present in said sample i) to bind with said dye ii); and

c) detecting the dye ii) bound to any of said target in the sample i), thereby detecting the presence, location or quantity of said target.

218. The dye of claim 217, wherein said alkene or alkyne comprises from 1-18 carbons.

219. The dye of claim 218, wherein said alkene or alkyne comprises from 1-6 carbons.

220. The method of claim 217, wherein said target comprises an oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, organic molecule, or a derivative thereof.

221. The method of claim 217, wherein said target has been bound to a solid matrix.

222. The method of claim 221, wherein said solid matrix comprises a bead, filter, gel, cuvette, test tube, dish, microscope slide, plate, microtitre plate or microarray.

223. The method of claim 222, wherein said target is detected in situ.

224. The method of claim 217, wherein said detection takes place without binding of target to a solid matrix.

225. The method of claim 224, wherein said detection takes place by a change in properties of said dye after binding takes place between said dye and said target.

226. The method of claim 225, wherein said change is an increase or decrease in the amount of fluorescence of said dye.

227. The method of claim 221, wherein a further step is carried out where unbound dye is removed.

228-773. -773. (canceled)

774. A process for increasing the amount of amplification of a target sequence, said process comprising amplifying said target sequence in the presence of an intercalating compound at a concentration where the efficiency of amplification is increased compared to the efficiency of amplification in the absence of said intercalating compound.

778. The process of claims 774, 775, 776 or 777, wherein said intercalating compound comprises an intercalating dye.

779. The process of claim 778, wherein said intercalating dye comprises SYBR Green I, Dye 109, Dye 110 or Dye 122.

780. The process of claims 774, 775, 776 or 777, wherein the amplified target sequences are detected after the conclusion of amplification.

781. The process of claim 778, wherein the amplified target sequences are detected during the course of amplification.

782. The process of claims 774, 775, 776 or 777, wherein said amplification is isothermal.

783. The process of claims 774, 775, 776 or 777, wherein said amplification is carried out by thermal cycling.

775. A process for increasing the specificity of amplification of a target sequence, said process comprising amplifying said target sequence in the presence of an intercalating compound at a concentration where the specificity of amplification is increased compared to the specificity of amplification in the absence of said intercalating compound.

776. A process for increasing the amount of amplification of a target sequence and for increasing the specificity of amplification of a target sequence, said process comprising amplifying said target sequence in the presence of an intercalating compound at a concentration where (i) the efficiency of amplification is increased compared to the efficiency of said intercalating compound, and (ii) the specificity of amplification is increased compared to the specificity of amplification in the absence of said intercalating compound.

777. A process for decreasing the amount of non-target amplification during a target amplification reaction, said process comprising amplifying said target sequence in the presence of an intercalating compound at a concentration where the efficiency of amplification of a non-target sequence is decreased compared to the efficiency of amplification of a non-target sequence in the absence of said intercalating compound.

784. A composition comprising at least a first portion and a second portion, wherein said first portion has an affinity for a first target and said second portion has an affinity for a second target, wherein said first target and said second target are different in nature and wherein said second portion has increased fluorescence upon binding to said second target.

785. A composite dye comprising a first component dye and a second component dye, wherein said first component dye has an affinity for a first target and said second component dye has an affinity for a second target, and wherein said composite has an affinity for a complex comprising said first target and said second target characterized in that said composite affinity is greater than the affinity of said first component dye for said first target or the affinity of said second component for said second target.

786. A composite dye comprising a first component dye and a second component dye, wherein said first component dye has an affinity for a first target and said second component dye has an affinity for a second target, wherein binding of said composite dye to a complex of said first target and said second target generates a signal with spectral characteristics that are different from a signal generated by binding of either said first component dye to said first target or by binding of said second component dye to said second target.