Compounds for use in PCR systems and applications thereof

Patent metadata

Field	Value
Publication number	US-9914964-B2
Application number	US-201414523410-A
Country	US
Kind code	B2
Filing date	Oct 24, 2014
Priority date	Oct 25, 2013
Publication date	Mar 13, 2018
Grant date	Mar 13, 2018

Abstract

This disclosure relates to novel compounds for use in various compositions, kits and methods, including, for example, use in polymerase storage buffers and in nucleic acid synthesis or amplification reactions such as a polymerase chain reaction (PCR). Methods for preparing the novel compounds are also described.

First claim

What is claimed is:

1. A composition comprising a polymerase and a compound having the structure:

wherein n is independently any positive integer, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

2. The composition of claim 1 , wherein said polymerase is thermostable.

3. The composition of claim 1 , further comprising one or more of the following:
a) at least one nucleotide triphosphate
b) DTT
c) KCl
d) MgCl 2
e) Kathon
f) EDTA
g) gelatin
h) Tris
i) glycerol
j) BSA.

4. The composition of claim 1 , further comprising a polymerase inhibitor.

5. The composition of claim 4 , wherein said polymerase inhibitor is at least one oligonucleotide inhibitor.

6. The composition of claim 4 , wherein said polymerase inhibitor is at least one antibody inhibitor.

7. The composition of claim 1 , wherein said polymerase is stable for at least about one month to about three years.

8. A kit comprising the composition of claim 1 .

9. The kit of claim 8 , wherein said polymerase is thermostable.

10. The kit of claim 9 , wherein said polymerase is a polymerase isolated from Thermus aquaticus (Taq) polymerase.

11. The kit of claim 9 , further comprising a polymerase inhibitor.

12. A method for increasing the efficiency of a polymerase, said method comprising the steps of:
a) mixing a target nucleic acid with at least one primer, dNTPs, and the composition of claim 1 ; and,
b) amplifying said target nucleic acid.

13. The method of claim 12 , wherein said increased polymerase efficiency is at least the same as the polymerase efficiency observed when said composition comprises NP-40 and/or Tween® 20 instead of said compound.

14. The method of claim 12 , wherein said increased polymerase efficiency is greater than the polymerase efficiency observed when the said composition comprises NP-40 and/or Tween® 20 instead of said compound.

15. The method of claim 12 , wherein the concentration of said compound is at least one to ten times less than the concentration of said NP-40 and/or Tween® 20.

16. A method for detecting a target nucleic acid in a sample, said method comprising the steps of:
a) forming a reaction mixture comprising at least one primer, dNTPs, the composition of claim 1 , and a detectable label;
b) amplifying said target nucleic acid; and,
c) detecting a signal generated from said detectable label indicative of the presence and/or amount of said target nucleic acid in said sample.

17. A nucleic acid amplification reaction mixture comprising:
a) the composition of claim 1 ; and
b) dNTPs.

18. The nucleic acid amplification reaction mixture of claim 17 , further comprising at least one primer.

19. The nucleic acid amplification reaction mixture of claim 17 , wherein said polymerase is thermostable.

20. The nucleic acid amplification reaction mixture of claim 17 , further comprising at least one antibody polymerase inhibitor.

21. The nucleic acid amplification reaction mixture of claim 17 , further comprising at least one oligonucleotide polymerase inhibitor.

22. A method for polymerizing a target nucleic acid comprising the steps of:
a) combining the target nucleic acid with the nucleic acid amplification reaction mixture of claim 17 ; and,
b) polymerizing the target nucleic acid.

23. A method for amplifying a target nucleic acid comprising the steps of:
a) combining the target nucleic acid with the nucleic acid amplification reaction mixture of claim 17 ; and,
b) amplifying the target nucleic acid.

24. The method of claim 22 or 23 wherein said reaction mixture further comprises at least one nucleic acid primer.

25. The method of claim 23 , wherein the amplified target nucleic acid is detected.

26. The method of claim 25 , wherein the amplified target nucleic acid is detected using a detectable label.

27. The method of claim 26 , wherein the detectable label is part of a primer or a probe.

28. The method of claim 23 , wherein the amplified target nucleic acid is quantitated.

29. The composition of claim 1 , wherein the polymerase is selected from the group consisting of T7 DNA polymerase, eukaryotic mitochondrial DNA Polymerase γ, prokaryotic DNA polymerase I, prokaryotic DNA polymerase II, prokaryotic DNA polymerase III, prokaryotic DNA polymerase IV, prokaryotic DNA polymerase V, eukaryotic polymerase α, eukaryotic polymerase β, eukaryotic polymerase γ, eukaryotic polymerase δ, eukaryotic polymerase ε, eukaryotic polymerase η, eukaryotic polymerase ζ, eukaryotic polymerase t, eukaryotic polymerase κ, E. coli DNA polymerase I, E. coli DNA polymerase III alpha subunit, E. coli DNA polymerase III epsilon subunits, E. coli polymerase IV, E. coli polymerase V, T. aquaticus DNA polymerase I, B. stearothermophilus DNA polymerase I, a Euryarchaeota polymerase, terminal deoxynucleotidyl transferase (TdT), S. cerevisiae polymerase 4, a translesion synthesis polymerase, reverse transcriptase, a thermostable polymerase, and telomerase.

30. The composition of claim 2 , wherein the thermostable polymerase is selected from the group consisting of Taq DNA polymerase, Tfi DNA polymerase, Tfl DNA polymerase, Pfu DNA polymerase, and Vent™ DNA polymerase, a polymerase having reduced 3′-to-5′ exonuclease activity, SuperScript™ DNA polymerase, a genetically engineered DNA polymerase, a polymerase having the active site mutation F667Y, a polymerase having the equivalent of active site F667Y, Tth polymerase, AmpliTaq®FS, ThermoSequenase™, Therminator I, Therminator II, Therminator III, Therminator Gamma, a derivative thereof, and a fragment thereof.

31. The composition of claim 2 , wherein the thermostable polymerase is Taq DNA polymerase.

32. The composition of claim 2 , wherein the thermostable polymerase is Tfl DNA polymerase.

33. A method for detecting a target nucleic acid in a sample, said method comprising:
a) forming a reaction mixture comprising at least one primer, dNTPs, and the composition of claim 1 , and a detectable label;
b) amplifying said target nucleic acid; and,
c) detecting a signal generated from said detectable label indicative of the presence of said target nucleic acid said sample.

34. The method of claim 33 , wherein said method comprises use of a hot start technique.

35. The method of claim 34 , wherein the hot start technique is selected from the group consisting of an oligonucleotide-based system, antibody-based system, chemical-based type system, and a dual hot start system.

Assignees

• Life Technologies Corp

Inventors

• Angrish Parul
• Yang Zhiwei

Classifications

• C07C303/28

  by reaction of hydroxy compounds with sulfonic acids or derivatives thereof · CPC title

• C07D211/60

  Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C07C211/63

  having quaternised nitrogen atoms bound to acyclic carbon atoms · CPC title