Nucleic acid binding dyes and uses therefor
View Patent ↗The invention provides novel compounds and compositions of Formulas I and II, as well as methods of using them. The compounds can be used, for example, to quantify an amount of double stranded DNA in a sample subjected to nucleic acid amplification, or for real time monitoring of a nucleic acid amplification reaction. The compounds can be provided in a kit, for example, with other reagents and instructions for using the compounds and reagents.
1. A method to quantify the amount of a nucleic acid in a sample subjected to nucleic acid amplification, comprising:
a) contacting the sample with a compound according to Formula 1:
wherein
R 1 , R 2 and R 3 are each H;
Y is S or O;
W taken together with the atoms to which it is attached is a membered heterocyclic ring;
n is 0;
Q is Q2:
wherein
Y is —CR 13 ═CR 14 —; m is 1 and p is 0;
R 6 is unsubstituted (C 1 -C 8 )alkyl, unsubstituted aryl, or unsubstituted (C 1 -C 8 )alkaryl;
R 9 , R 11 , and R 12 are each independently H, amino, or halo;
R 10 is H, methoxy, amino or halo;
one of R 13 and R 14 is H and the other is NR 15 R 16 ;
R 15 is (C 1 -C 6 )alkyl or (C 1 -C 6 )alkyl-NR 17 R 18 or C 3 H 7 —N + (CH 3 ) 3 , wherein R 17 and R 18 are each independently H or (C 1 -C 6 )alkyl; and
R 16 is (C 1 -C 6 )alkyl-NR 17 R 18 or C 3 H 7 —N + (CH 3 ) 3 , wherein R 17 and R 18 are each independently H or (C 1 -C 6 )alkyl;
or a salt thereof;
b) detecting fluorescence in the amplification reaction; and
c) correlating the amount of fluorescence in the amplification reaction with the amount of nucleic acid in the reaction.
2. The method of claim 1 further comprising determining the amount of template nucleic acid present prior to amplification.
3. The method of claim 1 wherein the compound does not substantially inhibit the amplification reaction.
4. The method of claim 1 wherein the amplification reaction further comprises a dye that does not have substantially altered fluorescence over a range of nucleic acid concentrations.
5. The method of claim 1 , wherein the nucleic acid is double-stranded DNA.
6. A method to quantify the amount of a nucleic acid in a sample subjected to nucleic acid amplification, comprising:
a) amplifying a nucleic acid in a sample, the sample comprising a target nucleic acid molecule, one or more nucleic acid primers specific for target sequences in the nucleic acid to be amplified, a polymerase, and a compound according to Formula I, so as to yield a mixture comprising amplified nucleic acid:
wherein
R 1 , R 2 and R 3 are each
Y is S or O;
W taken together with the atoms to which it is attached is a 6-membered heterocyclic ring;
n is 0;
Q is Q2:
wherein
Y is —CR 13 ═CR 14 —; m is 1 and p is 0;
R 6 is unsubstituted (C 1 -C 8 )alkyl, unsubstituted aryl, or unsubstituted (C 1 -C 8 )alkaryl;
R 9 , R 11 , and R 12 are each independently H, amino, or halo;
R 10 is H, methoxy, amino or halo;
one of R 13 and R 14 is H and the other is NR 15 R 16 ;
R 15 is (C 1 -C 6 )alkyl or (C 1 -C 6 )alkyl-NR 17 R 18 or C 3 H 7 —N + (CH 3 ) 3 , wherein R 17 and R 18 are each independently H or (C 1 -C 6 )alkyl; and
R 16 is (C 1 -C 6 )alkyl-NR 17 R 18 or C 3 H 7 —N + (CH 3 ) 3 , wherein R 17 and R 18 are each independently H or (C 1 -C 6 )alkyl;
or a salt thereof;
b) detecting fluorescence in the mixture; and
c) correlating the amount of fluorescence in the mixture with the amount of nucleic acid in the mixture.
7. The method of claim 6 wherein the target nucleic acid sequence is amplified using a polymerase chain reaction (PCR).
8. The method of claim 6 wherein the amount of fluorescence is correlated to the amount of target nucleic acid prior to amplification.
9. The method of claim 6 wherein the reaction mixture comprises a control dye for normalizing nucleic acid levels.
10. The method of claim 6 wherein the reaction mixture further comprises a known amount of internal control nucleic acid and primers specific therefore.
11. The method of claim 10 further comprising determining a ratio of amplification products of the target and control nucleic acid.
12. The method of claim 6 wherein amplification comprises thermally cycling the sample using initial predetermined temperature and time parameters.
13. The method of claim 12 wherein the reaction mixture is thermally cycled between at least a denaturation temperature and an elongation temperature.
14. The method of claim 6 wherein the nucleic acid is double-stranded DNA.
15. A method of real time monitoring of a nucleic acid amplification reaction, comprising
a) amplifying a target nucleic acid sequence in a target nucleic acid molecule in reaction mixture containing a sample having the target nucleic acid molecule, one or more nucleic acid primers specific for sequences in the target, and a compound according to Formula I:
wherein
R 1 , R 2 and R 3 are each H;
Y is S or O;
W taken together with the atoms to which it is attached is a 6-membered heterocyclic ring;
n is 0;
Q is Q2:
wherein
Y is —CR 13 ═CR 14 —; m is 1 and p is 0;
R 6 is unsubstituted (C 1 -C 8 )alkyl, unsubstituted aryl, or unsubstituted (C 1 -C 8 )alkaryl;
R 9 , R 11 , and R 12 are each independently H, amino, or halo;
R 10 is H, methoxy, amino or halo;
one of R 13 and R 14 is H and the other is NR 15 R 16 ;
R 15 is (C 1 -C 6 )alkyl or (C 1 -C 6 )alkyl-NR 17 R 18 or C 3 H 7 —N + CH 3 ) 3 , wherein R 17 and R 18 are each independently H or (C 1 -C 6 )alkyl; and
R 16 is (C 1 -C 6 )alkyl-NR 17 R 18 or C 3 H 7 —N + (CH 3 ) 3 , wherein R 17 and R 18 are each independently H or (C 1 -C 6 )alkyl;
or a salt thereof;
b) illuminating the reaction mixture with light of a selected wavelength that is absorbed by the compound; and
c) detecting the fluorescence emission in the reaction mixture.
16. The method of claim 15 wherein the target nucleic acid sequence is amplified using a polymerase chain reaction (PCR).