Apparatus for use in isothermal amplification
View Patent ↗An apparatus for monitoring amplification of a nucleic acid by providing a pH sensor or pH indicator, a nucleic acid in an amplification mixture, and an isothermal amplification enzyme.
1. An apparatus for monitoring isothermal amplification of a nucleic acid comprising:
a pH sensor or pH indicator;
a nucleic acid in an amplification mixture comprising a magnesium salt, an alkali base, and at least one of: a quaternary ammonium salt, ammonium chloride, or guanidine hydrochloride, wherein the mixture has a buffer capacity less than 10mM and is in communication with the pH sensor or pH indicator; and
an isothermal amplification enzyme.
2. The apparatus of claim 1 , wherein the pH indicator is a colorimetric or fluorescent dye.
3. The apparatus of claim 1 , wherein the pH sensor is an Ion Sensitive Field Effect Transistor (ISFET).
4. The apparatus of claim 1 , further comprising a reference electrode in fluid communication with the amplification mixture.
5. The apparatus of claim 1 , wherein the mixture further comprises one or more allele specific primers having at least one base complementary to a target Single Nucleotide Polymorphism (SNP) of the nucleic acid.
6. The apparatus of claim 1 , wherein the buffer capacity of the mixture is set to less than 5 mM.
7. The apparatus of claim 1 , wherein the buffer capacity of the mixture is set to less than 1 mM.
8. The apparatus of claim 1 , wherein the mixture further comprises a buffering agent.
9. The apparatus of claim 8 , wherein the buffering agent is selected from the group consisting of Tris, HEPES, bicine, and MOPS.
10. The apparatus of claim 9 , wherein the concentration of the buffering agent in the mixture is less than 5 mM.
11. The apparatus of claim 1 , wherein the mixture further comprises sulphate compounds.
12. The apparatus of claim 11 , wherein the concentration of the sulphate compounds in the mixture is less than 15 mM.
13. The apparatus of claim 1 , wherein the concentration of the quaternary ammonium salt, ammonium chloride, or guanidine hydrochloride in the mixture is between 2 mM and 15 mM.
14. The apparatus of claim 1 , wherein the concentration of the alkali base sets the pH of the mixture between 6 and 9.
15. The apparatus of claim 1 , wherein the alkali base is one of NaOH, KOH, or LiOH.
16. The apparatus of claim 1 , wherein the mixture further comprises one or more primers used in the amplification of the nucleic acid, which primers are allele specific such that amplification indicates the presence of a target nucleic acid.
17. The apparatus of claim 1 , wherein the isothermal amplification enzyme is a Strand Displacement polymerase.
18. The apparatus of claim 1 , wherein the isothermal amplification enzyme is a Loop-mediated isothermal amplification (LAMP) enzyme.
19. The apparatus of claim 1 further comprising a microfluidic structure configured to receive the amplification mixture.
20. The apparatus of claim 19 , wherein the microfluidic structure is exposed to the pH sensor.