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US 7,776,567 B2Granted
Dimeric and trimeric nucleic acid dyes, and associated systems and methods
AssigneesBiotium, Inc., AlleLogic Biosciences Corporation
InventorsFei Mao, Wai-Yee Leung, Xing Xin
Life of this patent
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Abstract
Dimeric and trimeric nucleic acid dyes, and associated systems and methods are provided. Such a dye may form a hairpin-like structure that enables it to stain nucleic acids via a release-on-demand mechanism, for example. Such a dye may have low background fluorescence in the absence of nucleic acids and high fluorescence in the presence of nucleic acids, upon binding therewith, for example. A dye provided herein may be useful in a variety of applications, such as in DNA quantitation in real-time PCR, for example.
Claims18
A method of performing a nucleic acid amplification reaction comprising:
- providing a reaction mixture comprising the target nucleic acid, reagents necessary for amplifying said target, and a fluorescent nucleic acid dye, the fluorescent nucleic acid dye having the formula:
- wherein BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 8 to about 150 non-hydrogen atoms;
- Q1 is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;
- Q2 is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;
- at least one dye constituent of the Q1 dye constituent and the Q2 dye constituent is a reporter dye constituent;
- at least one dye constituent of the Q1 dye constituent and the Q2 dye constituent is a fluorescent nucleic acid dye constituent or a non-fluorescent nucleic acid dye constituent;
- and the reporter dye constituent and the fluorescent nucleic acid dye constituent are optionally the same, wherein
- (i) when Q1 and/or Q2 is a phenanthridinium dye, at least one of Q1 and Q2 is a phenanthridinium dye has the structure of Formula I:
- R1 represents where BRIDGE attaches to the structure;
- and Ψ is an anion;
- or
- (ii) when each of Q1 and Q2 is an asymmetric cyanine dye, each of the Q1 and Q2 dye constituents has the structure of Formula II:
- wherein R1′ of Formula II is H;
- alkyl or alkenyl having 1 carbon to 6 carbons, inclusive;
- a halogen;
- —OR9;
- —SR10;
- —NR11R12;
- —CN;
- —NH(C═O)R13;
- —NHS(═O)2R14;
- —C(═O)NHR15;
- or a substituent associated with minor groove binding;
- or represents where BRIDGE attaches to the structure;
- when R1′ of Formula II comprises at least one of R9, R10, R11, R12, R13, R14 and R15, any said one of R9, R10, R11, R12, R13, R14 and R15, independently, is H or alkyl having 1 carbon to 12 carbons, inclusive, optionally incorporating 1 to 2 nitrogen(s), inclusive, or an aryl;
- when R1′ of Formula II comprises R11 and R12, R11 and R12 may in combination form a 5- or 6-membered saturated or unsaturated ring, which optionally comprises at least one hetero atom selected from N and O;
- X of Formula II is selected from O and S;
- n of Formula II is selected from 0, 1, and 2;
- R6 of Formula II is H;
- alkyl or alkenyl having 1 carbon to 10 carbons, inclusive, optionally comprising at least one hetero atom selected from N, O, and S;
- a halogen;
- —OR16;
- —SR16;
- —NR16R17;
- or a substituted or an unsubstituted aryl, optionally comprising 1 to 3 hetero atom(s), inclusive, selected from N, O, and S;
- or represents where BRIDGE attaches to the structure;
- R7 of Formula II is H;
- alkyl or alkenyl having 1 carbon to 10 carbons, inclusive, optionally comprising an aryl and at least one hetero atom selected from N, O, and S;
- or a substituted or an unsubstituted aryl optionally comprising 1 to 3 hetero atom(s), inclusive, selected from N, O, and S;
- or represents where BRIDGE attaches to the structure;
- R8 and R8′ of Formula II in combination form a fused aromatic ring, which may be further substituted 1 to 4 time(s), inclusive, independently, by C1-C2, inclusive, alkyl, C1-C2, inclusive, alkoxy, C1-C2, inclusive, alkylmercapto, or a halogen;
- each of R16 and R17 independently is H;
- alkyl having 1 carbon to 12 carbons, inclusive, optionally incorporating 1 to 2 nitrogen(s) or an aryl;
- or
- R16 and R17 may in combination form a 5- or 6-membered saturated or unsaturated ring, which optionally comprises at least one hetero atom selected from N and O;
- only one of R1′, R6 and R7 of Formula II represents where BRIDGE attaches to the structure;
- and
- Ψ of Formula II is an anion;
- or
- (iii) when either Q1 or Q2 is an acridine dye, at least one dye constituent of the Q1 and Q2 dye constituents has the structure of Formula III:
- wherein each R1 of Formula III is independently, is H or a C1-C2, inclusive, alkyl;
- one of R2 and R3 of Formula III represents where BRIDGE attaches to the structure;
- when R2 of Formula III represents where BRIDGE attaches to the structure, R3 is H or —CH3;
- when R3 of Formula III represents where BRIDGE attaches to the structure, R2 is selected from H, —CH3, —NH2, —NHCH3, —CN, and —C(═O)NH2;
- each R6 of Formula III independently, is H or a C1-C2, inclusive, alkyl;
- each R7 of Formula III independently, is H or a C1-C2, inclusive, alkyl;
- for each pair of adjacent R6 or R7 and R1, independently, R6 or R7 and R1 may in combination form a 5- or 6-membered, saturated or unsaturated ring;
- and
- Ψ of Formula III is an anion;
- subjecting said reaction mixture to a polymerization reaction under conditions suitable for formation of amplified target nucleic acids;
- illuminating the reaction mixture with light;
- and
- detecting fluorescence emission from said reaction mixture, wherein said emission is indicative of the presence of amplified target nucleic acids.
60 more elements
A method of performing a nucleic acid amplification reaction comprising:
- providing a reaction mixture comprising a target nucleic acid, reagents necessary for amplifying said target, and a fluorescent nucleic acid dye,
- the fluorescent nucleic acid dye having the formula:
- wherein BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 15 to about 150 non-hydrogen atoms;
- Q1 is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;
- Q2 is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;
- Q3 is a dye constituent selected from a fluorescent nucleic acid dye constituent, a non-fluorescent nucleic acid dye constituent, a fluorescent non-nucleic acid dye constituent, and a non-fluorescent non-nucleic acid dye constituent;
- at least one dye constituent of the Q1 dye constituent, the Q2 dye constituent, and the Q3 dye constituent is a reporter dye constituent;
- at least one dye constituent of the Q1 dye constituent, the Q2 dye constituent, and the Q3 dye constituent is a fluorescent nucleic acid dye constituent or a non-fluorescent nucleic acid dye constituent;
- and the reporter dye constituent and the fluorescent nucleic acid dye constituent are optionally the same;
- wherein, when one of Q1, Q2, or Q3 is an acridine dye, at least one dye constituent of the Q1, Q2 or Q3 dye constituents has the structure of Formula IV:
- wherein each R1 of Formula IV is independently, is H or a C1-C2, inclusive, alkyl;
- one of R2 and R3 of Formula IV represents where BRIDGE attaches to the structure;
- when R2 of Formula IV represents where BRIDGE attaches to the structure, R3 is H or —CH3;
- when R3 of Formula IV represents where BRIDGE attaches to the structure, R2 is selected from H, —CH3, —NH2, —NHCH2, —CN, and —C(═O)NH2;
- each R6 of Formula IV independently, is H or a C1-C2, inclusive, alkyl;
- each R7 of Formula IV independently, is H or a C1-C2, inclusive, alkyl;
- for each pair of adjacent R6 or R7 and R1, independently, R6 or R7 and R1 may in combination form a 5- or 6-membered, saturated or unsaturated ring;
- and
- Ψ of Formula IV is an anion;
- subjecting said reaction mixture to a polymerization reaction under conditions suitable for formation of amplified target nucleic acids;
- illuminating the reaction mixture with light;
- and
- detecting fluorescence emission from said reaction mixture, wherein said emission is indicative of the presence of amplified target nucleic acids.
20 more elements
Dependencies are inferred from the claim text.