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. An aqueous preparation comprising: a non-naturally occurring pH-sensitive dye, a DNA polymerase, dNTPs in a formulation that contains a weak buffering agent in an amount of less than 1 mM Tris or equivalent or no buffering agent.
2. A preparation according to claim 1 , further comprising primers.
3. A preparation according to claim 1 , further comprising a template DNA.
4. A preparation according to claim 1 , wherein the pH sensitive dye is either a visually detectable color dye or a fluorescent dye.
5. A method for detecting amplification of a nucleic acid; comprising:
providing an amplification reaction mixture comprising the aqueous preparation of claim 1 and a template DNA; and
detecting a change in spectral or fluorescent properties of the dye resulting from amplification of the target DNA.
6. The method according to claim 5 , wherein the amplification method comprises an isothermal nucleic acid amplification or a polymerase chain reaction.
7. A method according claim 6 , 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.
8. A method according to claim 7 , wherein the dye is soluble.
9. A method according to claim 8 , wherein the soluble dye is a colored dye detectable in visible light.
10. A method according to claim 9 , wherein the dye is selected from cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue, naphtolphthalein.
11. A method according to claim 8 , wherein the dye is a fluorescent dye.
12. A method according to claim 11 , wherein the fluorescent dye is a 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein or a carboxyl seminaphthorhodafluor.
13. A method according to claim 5 , wherein the weakly buffered solution comprises less than 1 mM of Tris buffer or equivalent buffer.
14. A method according to claim 5 , further comprising comparing a change in spectral or fluorescent properties of the pH sensitive dye from before to after the amplification reaction.