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Probes for detection of polymorphisms of c-kit gene, and use thereof

A technology of polymorphism detection and polymorphism, which is applied in the determination/inspection of microorganisms, material analysis through observation of the influence of chemical indicators, organic chemistry, etc., can solve problems such as costing a lot of labor and being impractical

Inactive Publication Date: 2012-10-03
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, in the detection method using such Tm analysis, for example, the difference of a single base must be judged by the Tm value
In addition, when a gene has multiple polymorphisms, it takes a lot of labor to analyze one sample
Therefore, there is also the problem of impracticality when analyzing multiple samples

Method used

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  • Probes for detection of polymorphisms of c-kit gene, and use thereof
  • Probes for detection of polymorphisms of c-kit gene, and use thereof
  • Probes for detection of polymorphisms of c-kit gene, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0221] In this example, Tm analysis was performed to detect the polymorphism of the c-kit gene under the condition that the wild type plasmid and the mutant plasmid coexisted.

[0222] As a partial sequence of the c-kit gene, a wild-type plasmid (WT) inserted with an oligonucleotide (SEQ ID NO: 23) having the D816 wild type and N822 wild type, and an oligonucleotide inserted with the D816V mutant type and the N822 wild type were prepared. D816V mutant plasmid (D816V) of oligonucleotide (SEQ ID NO: 24), N822K mutant plasmid (N822K) inserted with oligonucleotide (SEQ ID NO: 25) having D816 wild type and N822K mutant type. In the wild-type plasmid (WT), the base (w) of base number 108 in the base sequence shown in SEQ ID NO: 1 is adenine (a), and the base (d) of base number 127 is Thymine (t). In the D816V mutant plasmid (D816V), the base number 108 (w) in the base sequence shown in sequence number 1 is thymine (t), and the base number 127 (d) is thymine (t). In the N822K muta...

Embodiment 2

[0252] In this example, Tm analysis was performed in the coexistence of wild-type and mutant plasmids to detect the 816 polymorphism of the c-kit gene.

[0253] The D816V mutant plasmid (D816V) inserted with the oligonucleotides having the wild-type plasmid (WT) and the D816V mutant type described in Example 1 was mixed at a predetermined ratio as shown below to prepare three kinds of plasmid samples. For each 1 μL of plasmid sample, 2×10 4 copies / μL.

[0254] [Table 4]

[0255]

[0256] PCR and Tm analysis were performed in the same manner as in Example 1 except that 50 μL of the PCR reaction solution in Table 4 below and the following PCR and Tm analysis conditions were used. The PCR and Tm analysis conditions are as follows: 95°C for 1 second and 61°C for 15 seconds as a cycle, repeated 50 cycles after 95°C for 60 seconds, and 95°C for 1 second and 40°C for 60 seconds , and then set the rate of temperature rise to 1° C. / 3 seconds, heating from 40° C. to 75° C., and a ...

Embodiment 3

[0274] In this example, Tm analysis was performed to detect the 822 polymorphism of the c-kit gene under the condition that the wild-type plasmid and the mutant plasmid co-existed.

[0275] The N822K mutant plasmid (N822K) inserted with the oligonucleotides having the wild-type plasmid (WT) and the N822K mutant type described in Example 1 was mixed at a predetermined ratio shown below to prepare three kinds of plasmid samples. 1 × 10 per 1 μL of each plasmid sample 4 copies / μL.

[0276] [Table 6]

[0277]

[0278]

[0279] PCR and Tm analysis were performed in the same manner as in Example 2, except that the above-mentioned plasmid samples, the primers and probes shown below, and the following PCR conditions were used. The PCR conditions are 95°C for 60 seconds, 95°C for 1 second and 63°C for 15 seconds as a cycle, repeating 50 cycles, then 95°C for 1 second, and 40°C for 1 second. 60 seconds.

[0280] The sequences of the wild-type F primer and the mutant-type F pri...

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Abstract

The present invention provides probes (P1) to (P4) which can detect different polymorphisms of c-kit gene in a simple and highly reliable manner: (P1) an oligonucleotide which comprises a nucleotide sequence complementary to a nucleotide sequence having a 4- to 50-nucleotide length and containing nucleotide-105 to nucleotide-108 in SEQ ID NO:1 and which has a nucleotide complementary to nucleotide-105 at the 3'-terminal region; (P2) an oligonucleotide which comprises a nucleotide sequence having a 7- to 50-nucleotide length and containing nucleotide-102 to nucleotide-108 in SEQ ID NO:1 and which has a nucleotide corresponding to nucleotide-102 at the 5'-terminal region; (P3) an oligonucleotide which comprises a nucleotide sequence complementary to a nucleotide sequence having a 8- to 50-nucleotide length and containing a nucleotide-127 to nucleotide-134 in SEQ ID NO:1 and which has a nucleotide complementary to nucleotide-134 at the 5'-terminal region; and (P4) an oligonucleotide which comprises a nucleotide sequence having a 5- to 50-nucleotide length and containing nucleotide-123 to nucleotide-127 in SEQ ID NO:1 and which has a nucleotide corresponding to nucleotide-123 at the 5'-terminal region.

Description

technical field [0001] The present invention relates to a probe for detecting polymorphism of c-kit gene and its use. Background technique [0002] The c-kit receptor is a receptor-type tyrosine kinase on the cell surface, which binds to stem cell factors. The c-kit gene encoding the c-kit receptor is located on human chromosome 4. In the known c-kit gene, for example, in the partial sequence of the c-kit gene shown in SEQ ID NO: 1, the adenine (a) of base number 108 (w) is mutated into thymine (t), base Thymine (t) in base (d) of number 127 is mutated to guanine (g) or adenine (a) or the like (Non-Patent Document 1, etc.). In the c-kit receptor, by mutation of base number 108, aspartic acid (D) at position 816 is mutated to valine (V), and by mutation of base number 127, Asparagine (N) at position 822 was mutated to lysine (K). It has been reported that such mutations are associated with cancer diseases such as acute myeloid leukemia and gastrointestinal stromal tumor (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/68G01N21/78
CPCC12Q2600/156C07H21/04C12Q1/6886C12Q2527/101C12Q2527/107
Inventor 小森真理子井口亚希
Owner ARKRAY INC
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