Polymerization of nucleic acids using proteins having low isoelectric points

Patent metadata

Field	Value
Publication number	US-9567628-B2
Application number	US-201214124667-A
Country	US
Kind code	B2
Filing date	Jun 8, 2012
Priority date	Jun 8, 2011
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

Abstract

This disclosure relates to the use of one or more proteins (e.g., globular proteins) having a low isoelectric point and/or a limited number (e.g., zero) of modifying groups in nucleic acid polymerization and/or amplification reactions such as polymerase chain reaction (PCR).

First claim

What is claimed is:

1. A nucleic acid amplification reaction mixture comprising:
a) at least one polymerase;
b) dNTPs;
c) at least one protein having a low isoelectric point; and
d) a compound having the general formula:

wherein n is 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.

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

3. The reaction mixture of claim 1 , wherein said at least one protein having a low isoelectric point is BSA.

4. The reaction mixture of claim 1 , wherein said at least one protein having a low isoelectric point is selected from the group consisting of β-lactoglobulin, apomyoglobin, α-lactoalbumin, apotransferrin, α-acid glycoprotein, and β-casein.

5. The reaction mixture of claim 1 , wherein said at least one protein having a low isoelectric point is a negatively charged, globular protein when in a PCR reaction mixture.

6. The reaction mixture of claim 1 , wherein said at least one protein having a low isoelectric point has an isoelectric point less than 8 when in a PCR reaction mixture.

7. A method for increasing the efficiency of a polymerase, said method comprising:
a) mixing a target nucleic acid with at least one polymerase, at least one primer, dNTPs, at least one protein having a low isoelectric point, and a compound having the general formula:

wherein n is 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 the target nucleic acid.

8. The method of claim 7 , wherein the isoelectric point is between about 4 and about 7.

9. The method of claim 7 , wherein the isoelectric point is between about 4.2 to about 5.3.

10. The method of claim 7 , wherein the at least one protein is selected from the group consisting of α-acid glycoprotein, phosvitin, α-lactoalbumin, α-casein, β-casein, ovalbumin, bovine serum albumin, β-lactoglobulin, apotransferrin, and apomyoglobin.

11. The method of claim 7 , wherein the at least one protein is bovine serum albumin.

12. The method of claim 7 , wherein amplification of the target nucleic acid in step b) is detected.

13. The method of claim 12 , wherein amplification is detected using a detectable label.

14. The method of claim 13 , wherein the detectable label is part of a primer or probe.

15. The method of claim 12 , wherein the amplification is quantitated.

16. The method of claim 7 , 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 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.

17. The method of claim 16 , 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.

18. The method of claim 17 , wherein the thermostable polymerase is Taq DNA polymerase.

19. The method of claim 17 , wherein the thermostable polymerase is Tfi DNA polymerase.

Assignees

• Wang Jonathan
• Dupont David
• Life Technologies Corp

Inventors

• Wang Jonathan
• Dupont David

Classifications

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q1/6846

  Common amplification features · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title