Development of novel detergents for use in PCR systems

Patent metadata

Field	Value
Publication number	US-9493414-B2
Application number	US-201414250154-A
Country	US
Kind code	B2
Filing date	Apr 10, 2014
Priority date	Jun 8, 2011
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

Abstract

This disclosure relates to novel detergents for use in various procedures including, for example, nucleic acid amplification reactions such as polymerase chain reaction (PCR). Methods for preparing the modified detergents are also described.

First claim

What is claimed is:

1. 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 polymerase, at least one primer, at least one deoxyribonucleoside triphosphate (dNTP), and an effective concentration of at least one compound selected from:

wherein each n is independently 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, or 30; and
b) amplifying said target nucleic acid.

2. The method of claim 1 , wherein said increased efficiency is at least equivalent to the efficiency of said polymerase when said polymerase is in the presence of a nonionic detergent instead of said compound(s).

3. The method of claim 2 , wherein said increased efficiency is greater than the efficiency of said polymerase when said polymerase is in the presence of a nonionic detergent instead of said compound(s).

4. The method of claim 1 , wherein said effective concentration of said compound is at least one to ten times less than that required by a conventional detergent for increasing the efficiency of said polymerase.

5. The method of claim 4 , wherein said conventional detergent is a nonionic detergent.

6. The method of claim 4 , wherein said compound at said effective concentration is stable in a 5× buffer for at least 2 months.

7. The method of claim 1 , wherein said polymerase is thermostable.

8. A method for inhibiting inactivation of a polymerase in a thermal cycling process, said method comprising contacting said polymerase with at least one compound selected from:

wherein each n is independently 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, or 30,
during said thermal cycling process.

9. The method of claim 8 , wherein said inhibition of said polymerase inactivation is at least equivalent to the inhibition of said polymerase inactivation when said polymerase is in the presence of a nonionic detergent instead of said compound(s).

10. The method of claim 9 , wherein said inhibition of said polymerase inactivation is greater than the inhibition of polymerase inactivation when the polymerase is in the presence of a nonionic detergent instead of said compound(s).

11. The method of claim 8 , wherein said polymerase is thermostable.

12. A method for stabilizing polymerase activity of an enzyme, said method comprising combining an enzyme having polymerase activity and at least one compound selected from:

wherein each n is independently 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, or 30,
to form a mixture, under conditions such that said polymerase activity of said enzyme is stabilized.

13. The method of claim 12 , wherein said stabilization is at least equivalent to the stabilization of said polymerase activity when said polymerase is in the presence of a nonionic detergent instead of said compound(s).

14. The method of claim 13 , wherein said stabilization is greater than the stabilization of said polymerase activity when said polymerase is in the presence of a nonionic detergent instead of said compound(s).

15. The method of claim 12 , wherein said enzyme is thermostable.

16. A method for synthesizing a target nucleic acid comprising the steps of:
a) combining said target nucleic acid with at least one polymerase and at least one compound selected from:

wherein each n is independently 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, or 30,
as part of a reaction mixture; and
b) synthesizing said target nucleic acid.

17. The method of claim 16 , wherein said reaction mixture further comprises at least one nucleic acid primer and/or at least one deoxyribonucleoside triphosphate (dNTP).

18. The method of claim 16 , further comprising the step of:
c) detecting said target nucleic acid.

19. The method of claim 18 , wherein said detecting comprises the use of a detectable label.

20. The method of claim 19 , wherein said detectable label is part of a primer or a probe.

21. The method of claim 16 , further comprising the step of:
c) quantitating said target nucleic acid.

22. The method of claim 16 , wherein said 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 eukaryotic polymerase ζ, eukaryotic polymerase ι, 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.

23. The method of claim 22 , wherein said thermostable polymerase is selected from the group consisting of Taq DNA polymerase, Tfi DNA polymerase, Tfl DNA polymerase, Pfu DNA polymerase, a polymerase having reduced 3′-to-5′ exonuclease activity, a genetically engineered DNA polymerase, a polymerase having the active site mutation F667Y, a polymerase having the equivalent of active site F667Y, Tth polymerase, a derivative thereof, and a fragment thereof.

24. The method of claim 23 , wherein said thermostable polymerase is Taq DNA polymerase.

25. The method of claim 23 , wherein said thermostable polymerase is Tfl DNA polymerase.

Assignees

• Life Technologies Corp

Inventors

• Angrish Parul
• Yang Zhiwei
• Wang Jonathan

Classifications

• C07D207/16

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

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C07C229/12 Primary

  to carbon atoms of acyclic carbon skeletons · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C07C229/24

  having more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid · CPC title