Methods of using dyes in association with nucleic acid staining or detection and associated technology

A fluorescent nucleic acid dye having the formula:

• wherein BRIDGE is a substantially aliphatic linker comprising from about 8 to about 150 non-hydrogen atoms, inclusive, and wherein the linker comprises no more than one positive charge;
• Q1 is a fluorescent nucleic acid dye constituent;
• Q2 is a fluorescent nucleic acid dye constituent;
• and
• Q1 and Q2 may be the same or different, wherein
• (i) when Q1 and/or Q2 is a phenanthridinium dye, at least one of Q1 and Q2 is a phenanthridinium dye having 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.