Enzyme mixture

Patent metadata

Field	Value
Publication number	US-9617588-B2
Application number	US-201113252664-A
Country	US
Kind code	B2
Filing date	Oct 4, 2011
Priority date	Oct 5, 2010
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

Polymorphisms are present throughout an organism's genome, and understanding which alleles are present in a particular organism's genome can be advantageous. When probing the identity of these alleles, one must minimize incorrect readings due to inefficiencies in the system. In hydrolysis probe applications, these inefficiencies may be due to over-activity of an exonuclease functionality that excises nucleotides from probes that are only partially, complementary to a region of a target. The present invention provides a mixture that contains a plurality of polymerases including one that has a 5′→3′ exonuclease functionality and one that lacks or substantially lacks it, each in a sufficient relative amount and concentration to increase efficiencies of the system.

First claim

What is claimed is:

1. A composition comprising
a first DNA polymerase, wherein the first polymerase has a 5′→3′ exonuclease activity; and
a second DNA polymerase, wherein the second polymerase lacks or substantially lacks a 5′→3′ exonuclease activity, and
at least one hydrolysis probe comprising a quencher moiety and a reporter moiety, and optionally a 3′ modification,
the combined amount of first and second polymerases in the composition is between 100 polymerase activity units per milliliter and 500 polymerase activity units per milliliter, the ratio of the first polymerase to the second polymerase in the composition is 1:99 to 1:19.

2. The composition of claim 1 wherein the first polymerase is Tbr polymerase and the second polymerase is Tbr polymerase comprising a modification that deactivates or substantially deactivates 5′→3′ exonuclease activity.

3. The composition of claim 1 wherein the at least one reporter moiety is a fluorescent dye and the fluorescent dye on each of the hydrolysis probes exhibits distinguishable spectral properties, wherein the quencher is capable of quenching fluorescence of the fluorescent dye, and wherein the 3′ modification of the hydrolysis probes renders the hydrolysis probes less susceptible to 3′→5′ exonuclease degradation.

4. The composition of claim 1 wherein the hydrolysis probes anneals to different alleles of a gene, wherein the different alleles are alleles of a polymorphic gene.

5. The composition of claim 4 wherein the different alleles are single nucleotide polymorphisms.

6. The composition of claim 1 wherein the first polymerase, the second polymerase, or both the first and the second polymerase lacks or substantially lacks 3′→5′ exonuclease activity, or has less 3→5′ exonuclease activity than a wild type.

7. A method for detecting an allele of a target nucleic acid comprising
combining a target nucleic acid with a mixture of a first polymerase having a 5′→3′ exonuclease activity and a second polymerase lacking or substantially lacking a 5′→3′ exonuclease activity, where the ratio of the first polymerase to the second polymerase in the mixture is 1:99 to 1:19, and the combined amount of first polymerase and second polymerase is between 100 polymerase activity units per milliliter and 500 polymerase activity units per milliliter,
at least a first hydrolysis probe wherein the first probe is capable of annealing to a portion of the target nucleic acid containing a first form of an allele present in the target nucleic acid, and comprising a quencher moiety and a first reporter moiety,
a primer, wherein the primer is complementary to a sequence on the target nucleic acid upstream of the portion of the target nucleic acid complementary to the first probe, under conditions in which the primer can be extended, such that extension of the primer results in degradation of an annealed first probe and separation of the quencher moiety from the first reporter moiety, and resulting in a first signal from the first reporter moiety of the first probe; and
detecting the first signal from the first reporter moiety of the first probe, wherein detection of the first signal is indicative of the presence of the first form of the allele.

8. The method of claim 7 further comprising a second probe, wherein the second probe is capable of annealing to a portion of the target nucleic acid containing a second form of the allele present in the target nucleic acid, and comprising a quencher moiety and a second reporter moiety, and wherein degradation of an annealed second probe by extension of the upstream-annealing primer separates the quencher moiety from the second reporter moiety, resulting in a second signal from the second reporter moiety of the second probe; and detecting the second signal from the second reporter moiety of the second probe, which is distinguishable from the first signal, wherein detection of the second signal is indicative of the presence of the second form of the allele.

9. The method of claim 8 wherein a comparison of the first signal and the second signal allows for allelic discrimination of the target nucleic acid.

10. The method of claim 7 wherein the first polymerase is Tbr, and possesses a 5′→3′ exonuclease activity, and the second polymerase is Tbr, and lacks or substantially lacks a 5′→3′ exonuclease activity.

11. The method of claim 7 wherein the first polymerase, the second polymerase, or both the first and the second polymerase lacks or substantially lacks a 3′→5′ exonuclease activity, or has less 3′→5′ exonuclease activity than the wild type counterpart of the first polymerase, the second polymerase, or the first and second polymerases, respectively.

12. The method of claim 7 wherein at least one of the first polymerase or the second polymerase is a fusion polymerase.

13. The method of claim 12 wherein the fusion polymerase comprises a sequence-nonspecific double-stranded nucleic acid binding protein.

14. The method of claim 12 wherein the fusion polymerase comprises a sequence-nonspecific nucleic acid binding domain derived from Sac7d or Sso7d.

15. The method of claim 7 further comprising combining a reagent capable of creating a hot start condition with the polymerase mixture.

16. A kit comprising
a polymerase mixture, the mixture comprising at least a first DNA polymerase that possesses a 5′→3′ exonuclease activity and at least a second DNA polymerase that lacks or substantially lacks a 5′→3′ exonuclease activity, where the ratio of the first polymerase to the second polymerase in the mixture is 1:99 to 1:19, and the combined amount of first polymerase and second polymerase is between 100 polymerase activity units per milliliter and 500 polymerase activity units per milliliter, and
at least one hydrolysis probe comprising a quencher moiety and a reporter moiety, and optionally a 3′ modification.

17. The kit of claim 16 wherein the first polymerase is Tbr polymerase and the second polymerase is Tbr polymerase comprising a modification that deactivates or substantially deactivates 5′→3′ exonuclease activity.

18. A method for increasing the fidelity of allelic determination of a target nucleic acid using a dual-labeled probe, the method comprising
contacting the target nucleic acid with a mixture of a first polymerase having a 5′→3′ exonuclease activity and a second polymerase lacking or substantially lacking a 5′→3′ exonuclease activity, at least one hydrolysis probe, and at least one primer, where the ratio of the first polymerase to the second polymerase in the mixture is 1:99 to 1:19, and the combined amount of first polymerase and second polymerase is between 100 polymerase activity units per milliliter and 500 polymerase activity units per milliliter,
where the at least one probe is capable of annealing to a portion of the target nucleic acid containing the allele, and the probe comprises a quencher moiety and a reporter moiety,
where the primer is complementary to a sequence of the target nucleic acid which is upstream of the sequence to which the probe is complementary, under conditions in which the primer can be extended such that extension of the primer degrades an annealed probe and separates the quencher moiety from the reporter moiety; and
detecting a signal from the reporter moiety indicating the presence of the allele, where the method decreases the number of false positive results generated by a mis-annealed probe by balancing the levels of 5′→3′ exonuclease and polymerase activities in the mixture.

19. The method of claim 18 wherein the first polymeras

Assignees

• Kurkela Jaakko
• Thermo Fisher Scientific Baltics Uab

Inventors

• Kurkela Jaakko

Classifications

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q2535/131

  Allele specific probes · CPC title

• C12Q2561/101

  Taqman · CPC title