METHOD OF MEASURING PYROPHOSPHATE

• US 20090246783A1
• Filed: 02/27/2009
• Published: 10/01/2009
• Est. Priority Date: 02/28/2008
• Status: Active Grant

• Current Assignee: Asahi Kasei Pharma Corporation (Asahi Kasei Corporation), Ichibiki Co., Ltd.
• Original Assignee: Asahi Kasei Pharma Corporation (Asahi Kasei Corporation), Ichibiki Co., Ltd.
• Inventors: Imamura, Shigeyuki, Kumazawa, Toshihiko, SAKASEGAWA, Shinichi, Nishimura, Atsuhisa, Ikura, Yoshiaki
• Granted Patent: US 8,105,766 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/485 involving kinase

Abstract

Provided is a novel and useful method of measuring pyrophosphate in a sample. There may be provided a novel and useful method of measuring pyrophosphate in a sample using pyruvate orthophosphate dikinase, nicotinamide-nucleotide adenylyltransferase, and dehydrogenase.

Claims

1. A method of selectively measuring pyrophosphate in a sample, comprising the following steps (1) to (4):

(1) a step including a first reaction for producing ATP by bringing pyrophosphate which may be present in the sample into contact with pyruvate orthophosphate dikinase in the presence of at least AMP;

(2) a step including a second reaction for producing pyrophosphate and NADs by bringing the ATP produced by the first reaction into contact with nicotinamide-nucleotide adenylyltransferase;

(3) a step including a third reaction for reducing the NADs produced by the second reaction into NADHs; and

(4) a step of detecting the NADHs produced by the third reaction.

2. The method of selectively measuring pyrophosphate in a sample according to claim 1, wherein the step of detecting the NADHs produced by the third reaction includes the following steps:

(5) a step including a fourth reaction for converting the NADHs produced by the third reaction into a reduced nitroblue tetrazolium salt and NADs in the presence of a nitroblue tetrazolium salt; and

(6) a step of detecting the reduced nitroblue tetrazolium salt produced by the fourth reaction.

3. The method of selectively measuring pyrophosphate in a sample according to claim 2, wherein the NADs produced by the fourth reaction is used in the third reaction to perform a cycling reaction between the third reaction and the fourth reaction.

4. The method of selectively measuring pyrophosphate in a sample according to any one of claims 1 to 3, wherein the pyrophosphate produced by the second reaction is used in the first reaction to perform a cycling reaction between the first reaction and the second reaction.

5. The method of selectively measuring pyrophosphate in a sample according to claim 1, wherein the sample contains triphosphorylated deoxyribonucleic acids (dNTPs).

6. The method of selectively measuring pyrophosphate in a sample according to claim 1, wherein the nicotinamide-nucleotide adenylyltransferase is any of the following enzymes [1] to [3]:

[1] an enzyme which has an amino acid sequence of SEQ ID NO;

6 and may catalyze the second reaction;

[2] an enzyme which has the amino acid sequence of SEQ ID NO;

6 with deletion, substitution, or addition of one or several amino acids and may catalyze the second reaction; and

[3] an enzyme which has a substrate specificity ratio for dNTPs and ATP (substrate specificity for dNTPs/substrate specificity for ATP) of 5% or less and may catalyze the second reaction.

7. A method of detecting or quantifying a nucleic acid, comprising measuring, by the method according to claim 1, pyrophosphate in a sample subjected to nucleic acid amplification or a sample prepared from the sample.

11. A method for detecting or quantifying a nucleic acid, said method comprising measuring, by the method according to claim 1, pyrophosphate in a sample subjected to nucleic acid amplification or a sample prepared from the sample by using a composition comprising at least the following components (1) to (7):

(1) a metal ion;

(2) AMP;

(3) phosphoenolpyruvate;

(4) pyruvate orthophosphate dikinase;

(5) β

-nicotinamide mononucleotides;

(6) nicotinamide-nucleotide adenylyltransferase; and

(7) a reduced NAD substance.

12. A method for detecting or quantifying a nucleic acid, said method comprising measuring by the method according to claim 1, pyrophosphate in a sample subjected to nucleic acid amplification or a sample prepared from the sample by using a composition comprising the following two reagents (A) and (B):

(A) a first reagent containing at least the following components (1) to (4);

(1) a metal ion;

(2) AMP;

(3) phosphoenolpyruvate; and

(4) pyruvate orthophosphate dikinase;

(B) a second reagent containing at least the following components (5) to (7);

(5) β

-nicotinamide mononucleotides;

(6) nicotinamide-nucleotide adenylyltransferase; and

(7) a reduced NAD substance.

8. A composition comprising at least the following components (1) to (7):

(1) a metal ion;

(2) AMP;

(3) phosphoenolpyruvate;

(4) pyruvate orthophosphate dikinase;

(5) β

-nicotinamide mononucleotides;

(6) nicotinamide-nucleotide adenylyltransferase; and

(7) a reduced NAD substance.

10. The composition according to claim 8 or 9, wherein the nicotinamide-nucleotide adenylyltransferase is any of the following enzymes [1] to [3]:

[1] an enzyme which has an amino acid sequence of SEQ ID NO;

6 and may catalyze the second reaction;

[2] an enzyme which has the amino acid sequence of SEQ ID NO;

6 with deletion, substitution, or addition of one or several amino acids and may catalyze the second reaction; and

[3] an enzyme which has a substrate specificity ratio for dNTPs and ATP (substrate specificity for dNTPs/substrate specificity for ATP) of 5% or less and may catalyze the second reaction.

9. A composition comprising the following two reagents (A) and (B):

(A) a first reagent containing at least the following components (1) to (4);

(1) a metal ion;

(2) AMP;

(3) phosphoenolpyruvate; and

(4) pyruvate orthophosphate dikinase;

(B) a second reagent containing at least the following components (5) to (7);

(5) β

-nicotinamide mononucleotides;

(6) nicotinamide-nucleotide adenylyltransferase; and

(7) a reduced NAD substance.