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 determining cell viability, comprising:
a) contacting a sample comprising a cell 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 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;
wherein at least one of R15 and R16 is C 3 H 7 —N + (CH 3 ) 3 ;
or a salt thereof;
b) detecting a fluorescent signal in the sample; and
c) correlating the amount of fluorescence in the sample with the viability of the cell in the sample;
wherein an increase in the fluorescent signal correlates to a loss in cell membrane integrity.
2. The method of claim 1 , wherein the compound is impermeant to the cell membrane.
3. The method of claim 1 , wherein the loss in cell membrane integrity indicates cell death.
4. The method of claim 3 , wherein cell death is due to a cytotoxic effect of a test treatment or condition on the cell.
5. The method of claim 1 , further comprising a counterstain comprising a detectably different signal to correlate metabolically active cells.
6. The method of claim 5 , further comprising a counterstain comprising a detectably different signal to differentiate cells with a loss in cell membrane integrity and cells with intact cell membranes.
7. The method of claim 1 , wherein the compound is: