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Probe for detecting mutation in jak2 gene and use thereof

A probe and gene technology, applied in immunoassays, measurement devices, recombinant DNA technology, etc., can solve the problems of decreased detection sensitivity, difficult detection of high temperature side peaks, and difficulty in detection, and achieve the effect of good sensitivity

Active Publication Date: 2010-01-06
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this case, there is the following problem: in Tm analysis, when a melting curve representing the relationship between signal intensity and temperature is prepared, the high-temperature side peak belonging to the fully matched mutant sequence is due to the low-temperature side peak belonging to the mismatched normal sequence. exist but are difficult to detect
That is, even if there is a mutant sequence, its detection will become difficult due to the presence of the normal sequence, resulting in a decrease in detection sensitivity

Method used

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  • Probe for detecting mutation in jak2 gene and use thereof
  • Probe for detecting mutation in jak2 gene and use thereof
  • Probe for detecting mutation in jak2 gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Detection of the point mutation (g→t) of base 73 in exon 12 of JAK2 gene

[0098] Using the probe of the present invention, the point mutation (g→t) at base 79 of exon 12 of the JAK2 gene shown in SEQ ID NO: 1 was analyzed for Tm.

[0099] (test article)

[0100] JAK2 / V617F-positive leukemia cell line HEL and JAK2 wild-type leukemia cell line MYL were used. The culture solution of each cell was mixed so that the number of cells became the following ratio, and 10 μL of the culture mixture was treated at 95° C. for 5 minutes, and the treated sample was used as a sample for PCR.

[0101] [table 3]

[0102] HEL: MYL

(mt DNA) (wild-type DNA)

0:100

0 : 1×10 6 indivual

1:99

1×10 4 pcs: 99×10 4 indivual

10:90

1×10 5 pcs: 9×10 5 indivual

100:0

1×10 6 : 0

[0103] (probe and primer pair)

[0104] Detection probe (serial number 3)

[0105] 5'-agtatgtTtctgtggagac-(TAMRA)-3'

...

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Abstract

It is intended to provide a probe for detecting a mutation in JAK2 gene capable of detecting a sequence to be detected having a single nucleotide mutation even when the sequence to be detected having such a mutation and a sequence not to be detected having no mutation are present together. At least one oligonucleotide which comprises the same sequence as that of a region up to the 17th to the 22nd base from a first base toward the 5' direction when a cytosine base (C) at position 84 in exon 12 of JAK2 gene composed of a nucleotide sequence represented by SEQ ID NO: 1 is determined to be the first base, and has the cytosine base (C) as the 3' end is used as the probe. By using such a probe in, for example, Tm analysis, even if a sample contains JAK2 gene having a mutation and JAK2 gene having no mutation, the former mutation can be detected. The probe is preferably labeled with a fluorescent dye.

Description

technical field [0001] The present invention relates to a probe for detecting the mutation of JAK2 gene related to chronic myeloproliferative disorder (CMPD) and its use. Background technique [0002] As a method for analyzing the causes of all diseases at the genetic level, as well as the susceptibility of diseases among individuals (easiness of disease), differences in drug efficacy among individuals, etc., point mutations, that is, single nucleotide polymorphisms (SNPs) detection is widely used. [0003] Common detection methods for SNPs include: (1) direct sequencing (Direct Sequencing), which amplifies the region where the mutation of the detection target occurs and analyzes the base sequence of the resulting amplification product; (2) pyrosequencing (Pyrosequencing) method, (3) amplify the above-mentioned region, carry out HPLC in the temperature gradient column, detect the denaturing high performance liquid chromatography (Denaturing HPLC) method with or without muta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09G01N21/78
CPCC12Q2600/156C12Q1/6883Y10T436/143333G01N21/78
Inventor 平井光春间岛智史前川平木村晋也
Owner ARKRAY INC
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