Primer set for amplification of CYP2C9 gene, reagent for amplification of CYP2C9 gene comprising the same, and use of the same

A gene amplification and primer pair technology, applied in the field of reagents for CYP2C9 gene amplification, can solve the problems of reduced reliability of analysis results, inability to analyze a large number of samples, coding gene amplification, etc., and achieve rapid and simple amplification Reaction, rapid and simple analysis, shortening effect of amplification reaction

Inactive Publication Date: 2009-05-27
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in such a detection method using Tm analysis, there is a problem that in PCR, a region containing the detection target site must be amplified specifically and efficiently.
In particular, there are many isoenzymes of CYP, and the sequences encoding these enzymes are very similar, so there is a concern that even genes encoding isoenzymes other than CYP2C9 are amplified in PCR
Moreover, the fact that genes encoding other isozymes are also amplified also causes, for example, a reason for lowering the reliability of analysis results.
Furthermore, in this way, there is a problem that analysis of a large number of samples cannot actually be performed due to excessive labor involved in analyzing one sample.

Method used

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  • Primer set for amplification of CYP2C9 gene, reagent for amplification of CYP2C9 gene comprising the same, and use of the same
  • Primer set for amplification of CYP2C9 gene, reagent for amplification of CYP2C9 gene comprising the same, and use of the same
  • Primer set for amplification of CYP2C9 gene, reagent for amplification of CYP2C9 gene comprising the same, and use of the same

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Experimental program
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Effect test

preparation example Construction

[0045]

[0046] The method for preparing an amplification product of the present invention, as described above, is characterized in that it is a method for preparing an amplification product of a CYP2C9 gene by a gene amplification method, and includes the following step (I).

[0047] (1) A step of amplifying the above-mentioned CYP2C9 gene in a reaction solution using the nucleic acid in the sample as a template and using the CYP2C9 gene amplification primer pair of the present invention

[0048] As described above, by carrying out the amplification reaction using the primer pair of the present invention, it is possible to specifically and efficiently amplify the target region of the CYP2C9 gene including the detection target site of the polymorphism CYP2C9*3 as described above. Furthermore, the method for producing an amplification product of the present invention is characterized by using the primer pair of the present invention, and there are no limitations on the type, c...

Embodiment 1

[0113]Blood was collected from 9 test subjects using lithium heparin blood collection tubes (samples 1 to 9). 10 μL of the obtained blood was mixed with 90 μL of distilled water, and 10 μL of this mixture was mixed with 90 μL of distilled water. 10 μL of this mixture was added to 40 μL of a PCR reaction solution having the following composition, and PCR was performed using a thermal cycler. The PCR conditions were 95°C for 60 seconds, followed by 50 cycles of 95°C for 1 second and 52°C for 10 seconds, followed by 95°C for 1 second and 40°C for 60 seconds. Next, the rate of temperature rise was set at 1°C / 3 seconds, the PCR reaction solution was heated from 40°C to 95°C, and the change in fluorescence intensity over time was measured. The detection wavelength is 515-555nm (detection of fluorescent dye BODIPY FL). Also, the time required for 50 cycles of PCR is about 1 hour.

[0114] Table 4

[0115]

[0116]

[0117] * Product name Gene Taq FP: manufactured by Nippon...

Embodiment 2

[0129] Blood was collected from 2 test subjects using EDTA blood collection tubes (sample 1-2). 10 µL of the obtained blood was mixed with 70 µL of the diluent A described below, and 10 µL of this mixture was mixed with 70 µL of the diluent B described below. 10 μL of the obtained mixture was heat-treated at 95° C. for 5 minutes, then added to 46 μL of a PCR reaction solution with the following composition, and PCR was performed using a thermal cycler. The PCR conditions were 95°C for 60 seconds, followed by 50 cycles of 95°C for 1 second and 58°C for 15 seconds, followed by 95°C for 1 second and 40°C for 60 seconds. Next, the rate of temperature increase was set at 1°C / 3 seconds, the PCR reaction solution was heated from 40°C to 75°C, and the change in fluorescence intensity over time was measured. The detection wavelength is 515-555nm (detection of fluorescent dye BODIPYFL).

[0130] (Diluent A)

[0131] 10mM Tris-HCl (pH8), 0.1mM EDTA, 0.05% NaN 3 , 0.3% SDS

[0132] (...

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Abstract

Disclosed is a primer set for amplifying a region in CYP2C9 gene that contains a detection target site having CYP2C9*3 therein by a gene amplification method, which enables to amplify the region specifically. A pair of primer set is used, which comprises a forward primer comprising a nucleotide sequence depicted in SEQ ID NO:4 and a reverse primer comprising a nucleotide sequence depicted in SEQ ID NO:17. The primer set enables to amplify a region containing a site having a polymorphism CYP2C9*3 occurring in CYP2C9 gene in a specific manner and with a high degree of efficiency.

Description

technical field [0001] The present invention relates to a pair of primers for amplifying CYP2C9 gene, a reagent for amplifying CYP2C9 gene containing the pair of primers and an application thereof. Background technique [0002] Cytochrome P450 is an enzyme classified into a superfamily, and there are multiple subfamilies (for example, CYP1A, CYP1B, CYP2C, CYP2D, CYP2E, CYP3A, etc.). Among them, it has been elucidated that the mutation of the gene encoding the isoenzyme CYP2C9 of the human CYP2C subfamily (CYP2C9 gene) has an effect on the disposition and influence of the substrate drugs of CYP2C9-phenytoin (antiepileptic agent) and warfarin (anticoagulant drug). The effect has an impact. The polymorphism CYP2C9*3 of the CYP2C9 gene is known to be a mutation from isoleucine to leucine at amino acid position 359 (exon 7) (Non-Patent Document 1). In addition, the 359th amino acid position of CYP2C9*3 is the recognition site of the substrate drug, and changes in the amino acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/68G01N21/78
CPCC12Q2600/156C12Q1/6883C12Q1/6876C12N15/09G01N21/78
Inventor 平井光春间岛智史
Owner ARKRAY INC
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