# US 2010/0167279 A1 · Unsymmetrical cyanine dyes for high resolution nucleic acid melting analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0167279 A1 - Document ID: US20100167279A1 - Status: Published application - Kind: A1 - Application number: 12/346,260 - Filed: 2008-12-30 - Published: 2010-07-01 - Earliest claimed: 2008-12-30 - Assignees: Canon U.S. Life Sciences, Inc. - Inventors: Weidong CAO - Web page: https://patentgrep.com/patent/US20100167279A1 ## CPC - C07D 473/00: Heterocyclic compounds containing purine ring systems - C07D 413/06: linked by a carbon chain containing only aliphatic carbon atoms - C07D 413/14: containing three or more hetero rings - C07D 417/06: linked by a carbon chain containing only aliphatic carbon atoms - C07D 417/14: containing three or more hetero rings - C09B 23/04: one >CH- group, e.g. cyanines, isocyanines, pseudocyanines - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) - C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P) ## Abstract The present invention relates to novel unsymmetrical cyanine dyes and to methods of performing nucleic acid analysis in the presence of such dyes. More specifically, the present invention relates to novel unsymmetrical cyanine dyes that have high affinity to double-stranded nucleic acids and that do not inhibit amplification reactions, particularly the polymerase chain reaction (PCR). ## Claims ### Claim 1 An unsymmetrical cyanine dye having the Formula I wherein A is wherein X is O or S; R1, R2, R3, R4, R5, R6 and R7 are independently hydrogen, halogen, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted polyalkenyl, optionally substituted alkynyl, optionally substituted polyalkynyl, optionally substituted thioalkyl, optionally substituted aminoalkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted arylalkyl; R1 and/or R2 carry one or more positive charges such that the sum of the positive charge(s) on R1 and R2 is 1, 2 or 3; and R3, and/or R4, and/or R5, and/or R6 and/or R7 carry one or more positive charges such that the sum of the positive charge(s) on R3, R4, R5, R6 and R7 is 1, 2 or 3. ### Claim 2 (depends on 1) The unsymmetrical cyanine dye of claim 1, wherein A is ### Claim 3 (depends on 1) The unsymmetrical cyanine dye of claim 1, wherein A is ### Claim 4 (depends on 2) The unsymmetrical cyanine dye of claim 2, wherein R2 is hydrogen, alkyl, thiol, alkyl substituted with trialkylamino or alkyl substituted with alkoxy. ### Claim 5 (depends on 2) The unsymmetrical cyanine dye of claim 2, wherein R1 is aryl, alkyl substituted with dialkylamino, alkyl substituted with trialkylamino, alkyl substituted with alkoxy, alkyl substituted with alkoxy that is substituted with dialkylamino, or alkyl substituted with an N-substituted heterocyclyl. ### Claim 6 (depends on 2) The unsymmetrical cyanine dye of claim 2, wherein R1 is aryl, alkyl substituted with dialkylamino, alkyl substituted with trialkylamino, alkyl substituted with alkoxy, alkyl substituted with alkoxy that is substituted with dialkylamino, or alkyl substituted with an N-substituted heterocyclyl, and R2 is hydrogen, alkyl, thiol, alkyl substituted with trialkylamino or alkyl substituted with alkoxy. ### Claim 7 (depends on 3) The unsymmetrical cyanine dye of claim 3, wherein R3 is hydrogen, methyl, alkyl substituted with trialkylamino or alkyl substituted with an N-substituted heterocyclyl. ### Claim 8 (depends on 3) The unsymmetrical cyanine dye of claim 3, wherein R4 is hydrogen, alkyl, aryl, thiol, alkyl substituted with dialkyamino, alkyl substituted with trialkylamino, alkyl substituted with alkoxy that is substituted with dialkylamino, or alkyl substituted with an N-substituted heterocyclyl. ### Claim 9 (depends on 3) The unsymmetrical cyanine dye of claim 3, wherein R5 is hydrogen, halo, alkyl, aryl or alkyl substituted with alkoxy that is substituted with dialkylamino. ### Claim 10 (depends on 3) The unsymmetrical cyanine dye of claim 3, wherein R6 is hydrogen, alkyl, alkyl substituted with trialkylamino or alkyl substituted with an N-substituted heterocyclyl. ### Claim 11 (depends on 3) The unsymmetrical cyanine dye of claim 3, wherein R7 is hydrogen, alkyl or alkyl substituted with alkoxy. ### Claim 12 (depends on 3) The unsymmetrical cyanine dye of claim 3, wherein R3 is hydrogen, methyl, alkyl substituted with trialkylamino or alkyl substituted with an N-substituted heterocyclyl, R4 is hydrogen, alkyl, aryl, thiol, alkyl substituted with dialkyamino, alkyl substituted with trialkylamino, alkyl substituted with alkoxy that is substituted with dialkylamino, or alkyl substituted with an N-substituted heterocyclyl, R5 is hydrogen, halo, alkyl, aryl or alkyl substituted with alkoxy that is substituted with dialkylamino, R6 is hydrogen, alkyl, alkyl substituted with trialkylamino or alkyl substituted with an N-substituted heterocyclyl and R7 is hydrogen, alkyl or alkyl substituted with alkoxy. ### Claim 13 (depends on 1) The unsymmetrical cyanine dye of claim 1, wherein one or more positive charges is present in the native dye. ### Claim 14 (depends on 1) The unsymmetrical cyanine dye of claim 1, wherein one or more positive charges is present in the dye at a pH of an amplification reaction. ### Claim 15 (depends on 1) A method of nucleic acid analysis comprising the steps of: mixing an unsymmetrical cyanine dye of claim 1 with a sample comprising a target nucleic acid and one or more primers configured for amplifying the target nucleic acid, amplifying the target nucleic acid in the presence of the unsymmetrical cyanine dye, and monitoring fluorescence of the unsymmetrical cyanine dye. ### Claim 16 (depends on 15) The method of claim 15, wherein the monitoring step comprises melting the amplified target nucleic acid to generate a melting curve. ### Claim 17 (depends on 15) The method of claim 15, wherein the target nucleic acid comprises a single nucleotide polymorphism. ### Claim 18 (depends on 15) The method of claim 15 which further comprises identifying a genotype on the basis of the monitored fluorescence. ### Claim 19 (depends on 16) The method of claim 16 which further comprises identifying a genotype on the basis of the melting curve. ### Claim 20 (depends on 15) The method of claim 15, wherein the amplification and monitoring occur in a microfluidic channel. ### Claim 21 (depends on 15) The method of claim 15, wherein the monitoring step occurs during the amplification step. ### Claim 22 (depends on 15) The method of claim 15, wherein the monitoring step occurs subsequent to the amplification step. ### Claim 23 (depends on 15) The method of claim 15, wherein the monitoring occurs during the amplification step and subsequent to the amplification step. ### Claim 24 (depends on 23) The method of claim 23, wherein the monitoring step subsequent to the amplification step comprises melting the amplified target nucleic acid to generate a melting curve.