Nucleic acid amplification using a reversibly inactivated thermostable enzyme

• US 5,773,258 A
• Filed: 07/11/1996
• Issued: 06/30/1998
• Est. Priority Date: 08/25/1995
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Birch, David Edward, Laird, Walter Joseph, Zoccoli, Michael Anthony
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/680,283
• Time in Patent Office: 719 Days
• Field of Search: 435/91.2, 435/810, 435/183, 435/184
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/99 Enzyme inactivation by chemical treatment
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2549/101 Hot start
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention provides methods for the amplification of nucleic acids using a reversibly inactivated thermostable enzyme. The reversibly inactivated enzyme is the result of a chemical modification of the protein which inactivates the enzyme. The activity of the inactivated enzyme is recovered by an incubation of the reaction mixture at an elevated temperature prior to, or as part of, the amplification reaction. Non-specific amplification is reduced because the reaction mixture does not support the formation of extension products prior to the activating incubation.

Claims

1. A method for the amplification of a target nucleic acid contained in a sample comprising the steps of:

(a) contacting said sample with an amplification reaction mixture containing a primer complementary to said target nucleic acid and a modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable enzyme which catalyzes a primer extension reaction and a modifier reagent, wherein said reaction results in a covalent chemical modification of said enzyme which results in essentially complete inactivation of enzyme activity, wherein incubation of said modified enzyme in an aqueous buffer at alkaline pH at a temperature less than about 25°

C. results in no significant increase in enzyme activity in less than about 20 minutes, and wherein incubation of said modified enzyme in an aqueous buffer, formulated to about pH 8-9 at 25°

C., at a temperature greater than about 50°

C. results in at least a two-fold increase in enzyme activity in less than about 20 minutes; and

(b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50°

C. for a time sufficient to reactivate said enzyme and allow formation of primer extension products.

2. A method for the amplification of a target nucleic acid contained in a sample comprising the steps of:

(a) contacting said sample with an amplification reaction mixture containing a primer complementary to said target nucleic acid and a modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable ligase and a modifier reagent, wherein said reaction is carried out at alkaline pH at a temperature which is less than about 25°

C., wherein said reagent is a dicarboxylic acid anyhydride of the general formula;

##STR7## where R₁ and R₂ are hydrogen or organic radicals, which may be linked, or of the general formula;

##STR8## where R₁ and R₂ are organic radicals, which may be linked, and the hydrogen are cis and wherein said reaction results in essentially complete inactivation of enzyme activity; and

(b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50°

C. for a time sufficient to reactivate said enzyme and allow formation of primer extension products.

3. The method of claim 2, wherein said modifier reagent is selected from the group consisting of maleic anhydride;

exo-cis-3,6-endoxo-Δ

⁴ -tetrahydropthalic anhydride;

citraconic anhydride;

3,4,5,6-tetrahydrophthalic anhydride;

cis-aconitic anhydride; and

2,3-dimethylmaleic anhydride.

4. The method of claim 3, wherein said modifier reagent is citraconic anhydride or cis-aconitic anhydride.

5. The method of claim 4, wherein said wherein said modified thermostable enzyme is produced by a reaction of a mixture of said thermostable ligase and a greater than 20-fold molar excess of said compound.

6. A modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable enzyme which catalyzes a primer extension reaction and a modifier reagent, wherein said reaction results in a covalent chemical modification of said enzyme which results in essentially complete inactivation of enzyme activity, wherein incubation of said modified enzyme in an aqueous buffer at alkaline pH at a temperature less than about 25°

C. results in no significant increase in enzyme activity in less than about 20 minutes, and wherein incubation of said modified enzyme in an aqueous buffer, formulated to about pH 8-9 at 25°

C., at a temperature greater than about 50°

C. results in at least a two-fold increase in enzyme activity in less than about 20 minutes.

7. A modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable ligase and a modifier reagent, wherein said reaction is carried out at alkaline pH at a temperature which is less than about 25°

C., wherein said reagent is a dicarboxylic acid anyhydride of the general formula;

##STR9## where R₁ and R₂ are hydrogen or organic radicals, which may be linked, or of the general formula;

##STR10## where R₁ and R₂ are organic radicals, which may be linked, and the hydrogen are cis and wherein said reaction results in essentially complete inactivation of enzyme activity.

8. The modified thermostable enzyme of claim 7, wherein said modifier reagent is selected from the group consisting of maleic anhydride;

exo-cis-3,6-endoxo-Δ

⁴ -tetrahydropthalic anhydride;

citraconic anhydride;

3,4,5,6-tetrahydrophthalic anhydride;

cis-aconitic anhydride; and

2,3-dimethylmaleic anhydride.

9. The modified thermostable enzyme of claim 8, wherein said modifier reagent is citraconic anhydride or cis-aconitic anhydride.

10. The modified thermostable enzyme of claim 9, wherein said wherein said modified thermostable enzyme is produced by a reaction of a mixture of said thermostable ligase and a greater than 20-fold molar excess of said compound.

11. A kit for carrying out a polymerase chain reaction comprising a modified thermostable enzyme of claim 8.

12. A kit for carrying out a polymerase chain reaction comprising a modified thermostable enzyme of claim 9.

13. A kit for carrying out a polymerase chain reaction comprising a modified thermostable enzyme of claim 10.