Quencher and reporter dye combinations

Patent metadata

Field	Value
Publication number	US-11535885-B2
Application number	US-201915733557-A
Country	US
Kind code	B2
Filing date	Mar 1, 2019
Priority date	Mar 2, 2018
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

Disclosed is a probe for use in biological assays. The probe includes a fluorescent dye bound to a quencher compound through an oligonucleotide linker. Also disclosed are methods of using the probe, such as for a polymerase chain reaction (PCR), such as in a quantitative PCR reaction (qPCR), as well as kits including the probe.

First claim

The invention claimed is:

1. 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 R 4 , R 13 , and R 14 , and R 3 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 SO 3 , 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 R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , and R 22 is the same or different and is selected from the group consisting of H and SO 3 ;
each of R 2 and R 12 is the same or different and is selected from the group consisting of H and SO 3 ;
X is selected from the group consisting of —OH, —SH, —NH 2 , —NH—NH 2 , —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-NH 2 , —NR-L-NH—NH 2 , —NR-L-CO 2 H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NH(CH 2 CH 2 O) z CH 2 CH 2 N 3 , —NR-L-NH—CO—CH 2 —I, and an azide (N 3 )-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 + , Ca 2+ , 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 R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , and R 30 is the same or different and is independently selected from either H or SO 3 ;
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) an 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.

2. The probe of claim 1 , wherein:
R 5 , R 6 , R 9 , and R 10 are SO 3 ;
R 7 and R 8 are H;
R 4 , R 13 , and R 14 are methyl;
R 2 and R 12 are SO 3 ;
m, o, and p are 3; and n is 2.

3. The probe of claim 1 , wherein:
R 10 is SO 3 ;
R 5 , R 6 , R 7 , R 8 , and R 9 are H;
R 4 , R 13 , and R 14 are methyl;
R 12 is H;
R 2 is SO 3 ;
m and o are 3; and n and p are 2.

4. The probe of claim 1 , wherein:
R 10 is SO 3 ;
R 5 , R 6 , R 7 , R 8 , and R 9 are H;
R 4 , R 13 , and R 14 are methyl;
R 2 and R 12 are SO 3 ;
m, o, and p are 3; and n is 2.

5. The probe of claim 1 , wherein:
R 9 and R 10 are SO 3 ;
R 5 , R 6 , R 7 , and R 8 are H;
R 4 , R 13 , and R 14 are methyl;
R 12 is H;
R 2 is SO 3 ;
m and o are 3; and n and p are 2.

6. The probe of claim 1 , wherein:
R 9 and R 10 are SO 3 ;
R 5 , R 6 , R 7 , and R 8 are H;
R 4 , R 13 , and R 14 are methyl;
R 2 and R 12 are SO 3 ;
m, o, and p are 3; and n is 2.

7. The probe of claim 1 , wherein:
R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are H;
R 4 , R 13 , and R 14 are methyl;
R 2 and R 12 are H;
m and p are 1; n is 2; and o is 3.

8. The probe of claim 1 , wherein:
R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , and R 22 are H;
R 4 , R 13 , and R 14 are methyl;
R 2 and R 12 are H;
m and p are 1; n is 2; and o is 3.

9. The probe of claim 1 , wherein:
R 17 and R 18 are SO 3 ;
R 15 , R 16 , R 19 , R 20 , R 21 , and R 22 are H;
R 4 , R 13 , and R 14 are methyl;
R 2 and R 12 are SO 3 ;
m, o, and p are 3; and n is 2.

10. The probe of claim 1 , wherein:
R 5 , R 6 , R 9 , and R 10 are SO 3 ;
R 7 and R 8 are H;
R 4 , R 13 , and R 14 are methyl;
R 3 is —C-benzoate;
R 2 is SO 3 and Rig is H;
m is 3; p is 1; and n is 2.

11. The probe of claim 1 , wherein each of R 23 , R 25 , R 26 , R 27 , R 29 , and R 30 is H and each of R 24 and R 28 is SO 3 .

12. The probe of claim 1 , wherein each of R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , and R 30 is H.

13. The probe of claim 1 , wherein Z is NH-L, L being

and Y being the linkage to the solid support.

14. 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 R 4 , R 13 , and R 14 , and R 3 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 SO 3 , 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 R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , and R 22 is the same or different and is selected from the group consisting of H and SO 3 ;
each of R 2 and R 12 is the same or different and is selected from the group consisting of H and SO 3 ;
X is selected from the group consisting of —OH, —SH, —NH 2 , —NH—NH 2 , —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-NH 2 , —NR-L-NH—NH 2 , —NR-L-CO 2 H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, NH(CH 2 CH 2 O) z CH 2 CH 2 N 3 , —NR-L-NH—CO—CH 2 —I, and an azide (N 3 )-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 al

Assignees

• Life Technologies Corp

Inventors

• Mullah Khairuzzaman Bashar
• Evans Brian
• Benson Scott C
• Chang Chu-An
• Yan Xiongwei

Classifications

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title