Detection of an amplification reaction product using pH-sensitive dyes
View Patent ↗Methods are provided for a rapid, low cost approach to monitoring an amplification reaction. This includes monitoring the progress of isothermal or PCR amplification reactions to completion using pH-sensitive dyes that are either colored or fluorescent. Compositions are described that include a mixture of a DNA polymerase, deoxyribonucleotide triphosphate and a weak buffer of less than 1 mM Tris or equivalent or no buffer.
1. A method for detecting amplification of a nucleic acid; comprising:
providing an amplification reaction mixture containing a template DNA, nucleotides, a DNA polymerase and a non-naturally occurring pH-sensitive dye in a weakly-buffered or a non-buffered solution;
amplifying the template DNA; and
detecting a change in spectral or fluorescent properties of the dye resulting from amplification of the template DNA.
2. The method according to claim 1 , wherein the amplification method comprises an isothermal nucleic acid amplification or a polymerase chain reaction.
3. A method according claim 2 , wherein the isothermal nucleic acid amplification method is selected from the group consisting of a loop-mediated isothermal amplification, a helicase displacement amplification, a strand displacement amplification, a recombinase polymerase amplification and a nicking enzyme amplification reaction.
4. A method according to claim 3 , wherein the dye is soluble.
5. A method according to claim 4 , wherein the soluble dye is a colored dye detectable in visible light.
6. A method according to claim 5 , wherein the dye is selected from cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue, naphtolphthalein.
7. A method according to claim 4 , wherein the dye is a fluorescent dye.
8. A method according to claim 7 , wherein the fluorescent dye is a 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein or a carboxyl seminaphthorhodafluor.
9. A method according to claim 1 , wherein the weakly buffered solution comprises less than 1 mM of Tris buffer or equivalent buffer.
10. A method of monitoring a nucleic acid amplification of a target sequence if present in a sample, comprising: monitoring a change of pH in the presence of the target sequence as amplification proceeds beyond a threshold number of cycles, the monitoring being achieved by adding a non-naturally occurring pH-sensitive color or fluorescent dye to the reaction mixture in a weakly-buffered or non-buffered solution; amplifying the target sequence; and detecting a change in spectral or fluorescent properties of the dye after amplification compared to before amplification.