# US 7,745,135 B2 · Method for detecting reaction product of nucleic acid synthesis

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- Number: US 7,745,135 B2
- Document ID: US7745135B2
- Status: Granted
- Kind: B2
- Application number: 12/107,001
- Filed: 2008-04-21
- Published: 2009-03-26
- Granted: 2010-06-29
- Earliest claimed: 2000-05-01
- Assignees: Eiken Kagaku Kabushiki Kaisya
- Inventors: Yasuyoshi Mori; Kentaro Nagamine
- Web page: https://patentgrep.com/patent/US7745135B2

## CPC

- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for detecting the occurrence of nucleic acid syntheses using an enzyme through the use of a generated insoluble substance as an indicator.

## Claims

### Claim 1

A kit for detecting the occurrence of nucleic acid amplification or for monitoring nucleic acid amplification comprising the following elements:
(a) when a first arbitrary sequence F1c, a second arbitrary sequence F2c, and a third arbitrary sequence F3c are selected in that order from the 3′ terminus in a target region toward the 3′ terminus on the polynucleotide chain, and a fourth arbitrary sequence R1, a fifth arbitrary sequence R2, and a sixth arbitrary sequence R3 are selected in that order from the 5′ terminus in the target region toward the 5′ terminus of the nucleotide chain,
a primer containing sequence F2 which is complementary to F2c and, on the 5′ side of F2, the same sequence as F1c;
a primer containing sequence F3 which is complementary to F3c;
a primer containing the same sequence as R2 and, on the 5′ side of the sequence, sequence R1c which is complementary to R1; and
a primer containing the same sequence as R3;
(b) a polymerase catalyzing strand displacement-type synthesis of complementary chain;
(c) a nucleotide serving as a substrate for the element (b);
(d) a melting temperature regulator; and
(e) a coagulant.

### Claim 2

A kit for detecting the occurrence of nucleic acid amplification or for monitoring nucleic acid amplification comprising the following elements:
(a) when a first arbitrary sequence F1c and a second arbitrary sequence F2c are selected in that order from the 3′ terminus in a target region toward the 3′ terminus on the polynucleotide chain and a third arbitrary sequence R1 and a fourth arbitrary sequence R2 are selected in that order from the 5′ terminus in the target region toward the 5′ terminus of the nucleotide chain,
a primer containing sequence F2 which is complementary to F2c and, on the 5′ side of F2, the same sequence as F1c; and
a primer containing the same sequence as R2and, on the 5′ side of the sequence, sequence R1c which is complementary to R1;
(b) a polymerase catalyzing strand displacement-type synthesis of complementary chain;
(c) a nucleotide serving as a substrate for the element (b);
(d) a melting temperature regulator; and
(e) a coagulant.

### Claim 3 (depends on 1)

The kit according to claim 1, wherein the melting temperature regulator is any of betaine, trimethylamine N-oxide, proline, dimethylsulfoxide, or formamide, and the coagulant is polyacrylic acid or carboxymethyldextran.

### Claim 4 (depends on 2)

The kit according to claim 2, wherein the melting temperature regulator is any of betaine, trimethylamine N-oxide, proline, dimethylsulfoxide, or formamide, and the coagulant is polyacrylic acid or carboxymethyldextran.

### Claim 5 (depends on 1)

The kit according to claim 1, wherein the polymerase is selected from group consisting of Bst DNA polymerase, Bca(exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase I, Vent DNA polymerase, Vent(Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent(Exo-) DNA polymerase, φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, and KOD DNA polymerase.

### Claim 6 (depends on 2)

The kit according to claim 2, wherein the polymerase is selected from group consisting of Bst DNA polymerase, Bca(exo-) DNA polymerase, the Klenow fragment of E. coli DNA polymerase I, Vent DNA polymerase, Vent(Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent(Exo-) DNA polymerase, φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, and KOD DNA polymerase.