Nucleic acid binding dyes and uses therefor

Patent metadata

Field	Value
Publication number	US-9458499-B2
Application number	US-201514960589-A
Country	US
Kind code	B2
Filing date	Dec 7, 2015
Priority date	Mar 16, 2009
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

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.

First claim

What is claimed is:

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:

Assignees

• Promega Corp

Inventors

• Mcdougall Mark
• Dwight Stephen J

Classifications

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C07D513/06 Primary

  Peri-condensed systems · CPC title

• C09B23/04

  one >CH- group, e.g. cyanines, isocyanines, pseudocyanines · CPC title

• C12Q2563/107

  fluorescence · CPC title