Quencher and reporter dye combinations

A method of detecting or quantifying a target nucleic acid molecule in a sample by polymerase chain reaction (PCR), the method comprising:

• (i) contacting the sample comprising one or more target nucleic acid molecules with a) at least one probe having a sequence that is at least partially complementary to the target nucleic acid molecule, where the at least one probe undergoes a detectable change in fluorescence upon amplification of the one or more target nucleic acid molecules;
• and with b) at least one oligonucleotide primer pair;
• (ii) incubating the mixture of step (i) with a DNA polymerase under conditions sufficient to amplify one or more target nucleic acid molecules;
• and
• (iii) detecting the presence or absence or quantifying the amount of the amplified target nucleic acid molecules by measuring fluorescence of the probe, wherein the probe comprises a product of conjugation of:
• a) a dye having a general Formula Ia, Formula Ib, Formula Ic, or Formula Id
• wherein
• each of R4, R13, and R14, and R3 when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminal SO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, —F, —Cl, —Br, or a combination thereof;
• each of R5, R6, R7, R8, R9, R10, R15, R16, R17, R18, R19, R20, R21, and R22 is the same or different and is selected from the group consisting of H and SO3;
• each of R2 and R12 is the same or different and is selected from the group consisting of H and SO3;
• X is selected from the group consisting of —OH, —SH, —NH2, —NH—NH2, —F, —Cl, —Br, I, —O—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH2, —NR-L-NH—NH2, —NR-L-CO2H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, NH(CH2CH2O)zCH2CH2N3, —NR-L-NH—CO—CH2—I, and an azide (N3)-containing group, where R is —H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom;
• Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine;
• m is an integer from 0 to 5 inclusive;
• n is an integer from 1 to 3 inclusive;
• o is an integer from 0 to 12 inclusive;
• and p is an integer from 0 to 5 inclusive;
• b) a quencher having general Formula II
• wherein
• each of R23, R24, R25, R26, R27, R28, R29, and R30 is the same or different and is independently selected from either H or SO3;
• Z is OR, where R is alkyl, or Z is NH-L, where L is
• and Y is either H or a linkage to a solid support;
• and
• c) at least one oligonucleotide linker joining the dye and the quencher, wherein the dye is covalently joined to a first nucleotide of the oligonucleotide and the quencher is covalently joined to a second, different nucleotide of the oligonucleotide.

A kit for polymerase chain reaction (PCR), the kit comprising:

• a probe comprising a product of conjugation of:
• a) a dye having a general Formula Ia, Formula Ib, Formula Ic, or Formula Id
• wherein
• each of R4, R13, and R14, and R3 when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminal SO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, —F, —Cl, —Br, or a combination thereof;
• each of R5, R6, R7, R8, R9, R10, R15, R16, R17, R18, R19, R20, R21, and R22 is the same or different and is selected from the group consisting of H and SO3;
• each of R2 and R12 is the same or different and is selected from the group consisting of H and SO3;
• X is selected from the group consisting of —OH, —SH, —NH2, —NH—NH2, —F, —Cl, —Br, I, —O—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH2, —NR-L-NH—NH2, —NR-L-CO2H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, NH(CH2CH2O)zCH2CH2N3, —NR-L-NH—CO—CH2—I, and an azide (N3)-containing group, where R is —H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom;
• Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine;
• m is an integer from 0 to 5 inclusive;
• n is an integer from 1 to 3 inclusive;
• o is an integer from 0 to 12 inclusive;
• and p is an integer from 0 to 5 inclusive;
• b) a quencher having general Formula II
• wherein
• each of R23, R24, R25, R26, R27, R28, R29, and R30 is the same or different and is independently selected from either H or SO3;
• Z is OR, where R is alkyl, or Z is NH-L, where L is
• and Y is either H or a linkage to a solid support;
• and
• c) at least one oligonucleotide linker joining the dye and the quencher, wherein the dye is covalently joined to a first nucleotide of the oligonucleotide and the quencher is covalently joined to a second, different nucleotide of the oligonucleotide;
• and
• one or more of the following:
• a buffering agent, a purification medium, the target, an organic solvent, an enzyme, and an enzyme inhibitor.

A kit comprising at least one a dye having a general Formula Ia, Formula Ib, Formula Ic, or Formula Id

• wherein
• each of R4, R13, and R14, and R3 when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminal SO3, a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, —F, —Cl, —Br, or a combination thereof;
• each of R5, R6, R7, R8, R9, R10, R15, R16, R17, R18, R19, R20, R21, and R22 is the same or different and is selected from the group consisting of H and SO3;
• each of R2 and R12 is the same or different and is selected from the group consisting of H and SO3;
• X is selected from the group consisting of —OH, —SH, —NH2, —NH—NH2, —F, —Cl, —Br, I, —O—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH2, —NR-L-NH—NH2, —NR-L-CO2H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, NH(CH2CH2O)zCH2CH2N3, —NR-L-NH—CO—CH2—I, and an azide (N3)-containing group, where R is —H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom;
• Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine;
• m is an integer from 0 to 5 inclusive;
• n is an integer from 1 to 3 inclusive;
• o is an integer from 0 to 12 inclusive;
• and p is an integer from 0 to 5 inclusive;
• and
• at least one quencher having general Formula II
• wherein
• each of R23, R24, R25, R26, R27, R28, R29, and R30 is the same or different and is independently selected from either H or SO3;
• Z is OR, where R is alkyl, or Z is NH-L, where L is
• and Y is either H or a linkage to a solid support.