Bst DNA polymerase, reagent composition and nucleic acid amplification method

Abstract

The purpose of the present invention is to provide a Bst DNA polymerase that can reduce the effects of interfering substances and reactivity at temperatures lower than the optimum reaction temperature. In addition, the invention aims to provide a reagent composition containing the Bst DNA polymerase, a kit containing the Bst DNA polymerase, and a nucleic acid amplification method using the Bst DNA polymerase. The problem is solved by a Bst DNA polymerase which contains an amino acid sequence represented by SEQ ID NO: 1, or a consensus sequence Asn-X-(Ser / Thr) (X is any amino acid residue other than a proline residue), contains an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1, has 5 '-3' DNA polymerase activity and chain substitution activity, and has a consensus sequence containing an N-binding sugar chain, and the N-binding sugar chain contains more than three mannose residues.

Description

[Technical field] The present invention relates to a Bst DNA polymerase having an N-linked sugar chain. The present invention relates to a reagent composition for use in a nucleic acid amplification method, which contains the Bst DNA polymerase. The present invention relates to a nucleic acid amplification method using a Bst DNA polymerase having an N-linked sugar chain. [Background technology] Bst DNA polymerase is a heat-resistant DNA polymerase derived from Geobacillus stearothermophilus, having 5'→3' DNA polymerase activity and chain substitution activity. Since Bst DNA polymerase has chain substitution activity, isothermal nucleic acid amplification reaction can be performed. Isothermal nucleic acid amplification reaction is used in, for example, the fields of medicine, food hygiene, environmental hygiene, etc., in genetic testing of cancer, genetic testing for detecting viruses or bacteria, etc. Since the activity of Bst DNA polymerase becomes maximum at 60°C to 65°C, Bst DNA polymerase is also suitable for the synthesis of DNA chains with high GC content. In Patent Document 1, it is recorded that recombinant Bst DNA polymerase is produced by an Escherichia coli expression system. [Prior art literature] [Patent Literature] [Patent Document 1] U.S. Patent No. 5,814,506 [Summary of the Invention] [Problems to be solved by the invention] Generally speaking, it is known that in a nucleic acid amplification reaction, the amplification efficiency of the target nucleic acid is reduced by mixing an interfering substance into the reaction solution. An interfering substance is a substance that causes the inhibition of the nucleic acid amplification reaction, the reduction of the reaction speed, etc. The interfering substance has a sample containing the target nucleic acid, such as a biological sample contained in blood, cells, fixed embedded tissues, etc. The reagent used when extracting and purifying the target nucleic acid from such a sample can also become an interfering substance. It is also known that in a nucleic acid amplification reaction, at a temperature lower than the optimum reaction temperature of the DNA polymerase used, non-specific amplification of the nucleotide sequence that is not dependent on the target nucleic acid is prone to occur. For example, when the reaction solution is heated to the optimum reaction temperature, non-specific amplification products can occur before the optimum reaction temperature is reached. When using an isothermal nucleic acid amplification reaction using Bst DNA polymerase in the detection of the target nucleic acid, the influence of the interfering substance and the reactivity at a temperature lower than the optimum reaction temperature have concerns about reducing the detection sensitivity. The present invention aims to provide a Bst DNA polymerase that can reduce the influence of interfering substances and the reactivity at a temperature lower than the optimal reaction temperature. In addition, the present invention aims to provide a reagent composition containing the Bst DNA polymerase and a nucleic acid amplification method using the Bst DNA polymerase.

Methods for solving the problem

[21] .

[0001] A Bst DNA polymerase comprising (1) the amino acid sequence shown in SEQ ID NO: 1, or (2) the consensus sequence Asn-X-(Ser / Thr) (X is any amino acid residue other than a proline residue), and comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having 5'→3' DNA polymerase activity and strand substitution activity, wherein the consensus sequence comprises an N-linked sugar chain, and the N-linked sugar chain comprises three or more mannose residues.

[0002] [1] The Bst DNA polymerase described above, wherein the finger domain contains a consensus sequence.

[0003] The Bst DNA polymerase described in [1] or [2], wherein the N-linked sugar chain contains 9 or less mannose residues.

[0004] The Bst DNA polymerase according to any one of [1] to [3], wherein the N-linked sugar chain has a structure represented by the following formula (I):

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[0005] [4] The Bst DNA polymerase described above, wherein S1 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue and a glucose residue.

[0006] The Bst DNA polymerase described in [4] or [5], wherein S2 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue and a glucose residue.

[0007] The Bst DNA polymerase according to any one of [4] to [6], wherein S3 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue and a glucose residue.

[0008] The Bst DNA polymerase according to any one of [4] to [7], wherein S4 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue and a glucose residue.

[0009] [4] The Bst DNA polymerase described above, wherein S1, S2, S3 and S4 are not present.

[0010] The Bst DNA polymerase according to any one of [4] to [8], wherein at least one sugar chain of S1, S2, S3 and S4 is an N-acetyl D-glucosamine residue.

[0011] The Bst DNA polymerase according to any one of [4] to [8], wherein at least one sugar chain of S1, S2, S3 and S4 has a structure represented by the following formula (II):

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[0012] The Bst DNA polymerase according to any one of [1] to

[11] , wherein at least one non-reducing end of the N-linked sugar chain is a mannose residue or an N-acetyl D-glucosamine residue.

[0013] The Bst DNA polymerase described in [1] to

[12] , wherein m is 1 and the above n is 0, or the above m is 0 and the above n is 1.

[0014] A reagent composition for use in a nucleic acid amplification method, comprising the BstDNA polymerase described in any one of [1] to

[13] .

[0015]

[14] The reagent composition described in claim 14, wherein the nucleic acid amplification method is performed under substantially isothermal conditions.

[0016] The reagent composition described in

[14] or

[15] , wherein the nucleic acid amplification method is the LAMP method, the RCA method, the NASBA method, the SDA method, the TRC method or the ICAN method.

[0017] A kit for use in a nucleic acid amplification method, comprising the reagent composition described in any one of

[14] to

[16] and dNTPs.

[0018]

[17] The kit described in claim 1, wherein the nucleic acid amplification method is performed under substantially isothermal conditions.

[0019] The kit described in

[17] or

[18] , wherein the nucleic acid amplification method is LAMP method, RCA method, NASBA method, SDA method, TRC method or ICAN method.

[0020] A nucleic acid amplification method, comprising amplifying a target nucleic acid using the Bst DNA polymerase described in any one of [1] to

[13] .

[0021]

[20] The nucleic acid amplification method described above, wherein the amplification reaction of the target nucleic acid is carried out under substantially isothermal conditions. [Effects of the Invention] The present invention can provide a Bst DNA polymerase that can reduce the influence of interfering substances and the reactivity at a temperature lower than the optimal reaction temperature. In addition, the present invention can provide a reagent composition containing the Bst DNA polymerase and a nucleic acid amplification method using the Bst DNA polymerase. [Brief description of the attached drawings]

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Explanation of symbols

Claims

1. Bst DNA polymerase, comprising: (1) the amino acid sequence shown in SEQ ID NO: 1, or (2) consensus sequence Asn-X-(Ser / Thr), wherein X is any amino acid residue other than a proline residue, and The Bst DNA polymerase contains an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and has 5'→3' DNA polymerase activity and strand displacement activity, The consensus sequence contains an N-bound sugar chain, The N-linked sugar chain contains three or more mannose residues.

2. The Bst DNA polymerase of claim 1, wherein the consensus sequence is contained in the finger domain.

3. The Bst DNA polymerase of claim 1, wherein the N-bound sugar chain contains 9 or less mannose residues.

4. The Bst DNA polymerase according to claim 3, wherein the N-bound sugar chain has a structure represented by the following formula (I): 【Chemistry 1】 In the formula, Asn is the asparagine residue in the consensus sequence, Man is a mannose residue, GlcNAc is an N-acetyl D-glucosamine residue, Fuc is a fucose residue, m and n are each independently 0 or 1, S1, S2, S3 and S4 are each independently absent or contain a sugar chain of 1 to 5 monosaccharide residues. 5 . The Bst DNA polymerase of claim 4 , wherein the S1 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue, and a glucose residue.

6. The Bst DNA polymerase of claim 4, wherein the S2 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue, and a glucose residue.

7. The Bst DNA polymerase of claim 4, wherein the S3 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue, and a glucose residue.

8. The Bst DNA polymerase of claim 4, wherein the S4 contains at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl D-glucosamine residue, and a glucose residue.

9. The Bst DNA polymerase of claim 4, wherein the S1, the S2, the S3, and the S4 are absent. 10 . The Bst DNA polymerase of claim 4 , wherein at least one sugar chain of the S1, S2, S3, and S4 is an N-acetyl D-glucosamine residue.

11. The Bst DNA polymerase according to claim 4, wherein at least one sugar chain of the S1, S2, S3 and S4 has a structure represented by the following formula (II): 【Chemistry 2】 In the formula, S5 and S6 are each independently absent or a sugar chain containing 1 to 3 mannose residues. 12 . The Bst DNA polymerase of claim 1 , wherein at least one non-reducing end of the N-bound sugar chain is a mannose residue or an N-acetyl D-glucosamine residue.

13. The Bst DNA polymerase of claim 9, wherein The m is 1, and the n is 0, or The m is 0, and the n is 1.

14. A reagent composition for use in a nucleic acid amplification method, comprising the Bst DNA polymerase according to any one of claims 1 to 13.

15. The reagent composition of claim 14, wherein the nucleic acid amplification method is performed under substantially isothermal conditions. 16 . The reagent composition according to claim 15 , wherein the nucleic acid amplification method is a LAMP method, an RCA method, a NASBA method, an SDA method, a TRC method or an ICAN method.

17. A method for amplifying a nucleic acid, comprising amplifying a target nucleic acid using the Bst DNA polymerase according to any one of claims 1 to 13.

18. The nucleic acid amplification method according to claim 17, wherein the amplification reaction of the target nucleic acid is performed under substantially isothermal conditions.

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