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Primer, molecular beacon and kit for rapidly detecting CYP2C9*3 gene polymorphism and detection method of CYP2C9*3 gene polymorphism

A gene polymorphism and detection method technology, applied in the field of primers for rapid detection of CYP2C9*3 gene polymorphism, can solve the problems of reducing the comfort level of the test subject, pollution, and complicated operation

Inactive Publication Date: 2017-10-03
重庆京因生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invasive detection of the existing mode will not only reduce the detection comfort of the examinee, but also make it difficult to meet the rapid diagnosis of clinical diseases, increase the risk of disease treatment for patients, and the operation is complicated and the chance of contamination is high

Method used

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  • Primer, molecular beacon and kit for rapidly detecting CYP2C9*3 gene polymorphism and detection method of CYP2C9*3 gene polymorphism
  • Primer, molecular beacon and kit for rapidly detecting CYP2C9*3 gene polymorphism and detection method of CYP2C9*3 gene polymorphism
  • Primer, molecular beacon and kit for rapidly detecting CYP2C9*3 gene polymorphism and detection method of CYP2C9*3 gene polymorphism

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Experimental program
Comparison scheme
Effect test

Embodiment 3

[0058] (3) Preparation of 200X cell lysate in embodiment three

[0059] Use a 1.5mL centrifuge tube, add 300ul SDS and 56.34ul Triton X-100, then add 643.66ul nuclease-free water to a total volume of 1000ul, make the final concentration of SDS reach 3%, make the final concentration of TritonX-100 reach 6% , that is, 200x cell lysate, then shake and mix well, and store at -4°C.

[0060] The gene sequence of the forward primer 5'-3' is: CTGCATGCAAGACAGGA;

[0061] The gene sequence of the reverse primer 5'-3' is: AACTTACCTTGGGAATGAGA.

[0062] The gene sequence of wild-type probe 5'-3' is:

[0063] (Fam marker)-CGACGTGATA+C+A+TTGACCTTCTCCCACGTCG-(BHQ1 marker);

[0064] The gene sequence of the mutant probe 5'-3' is:

[0065] (Alexa Fluor 594 labeled)-CGCACGCAGAGATA+C+C+TTGACCTTCCGTGCG-(BHQ2 labeled);

[0066] + is the modified base of locked nucleic acid.

[0067] The above raw materials, except for specific primers, molecular beacons, and cell lysates, were purchased from...

Embodiment 1

[0087] The detection result of embodiment one is:

[0088] Such as figure 1 , figure 2 and image 3 As shown, line A in the figure indicates that CYP2C9*3 is not detected, and line C indicates that CYP2C9*3 is detected.

[0089] Depend on figure 1 It can be seen that only line A has exponential growth, but line C has no exponential growth, so the test result is wild homozygous AA, suggesting that the expression or activity of the detected gene may be normal, and this genotype is a normal metabolic type;

[0090] Depend on figure 2 It can be seen that both line A and line C have exponential growth, indicating that the test result is mutant heterozygous AC, suggesting that the activity of the detected gene may decrease, and this genotype belongs to the moderate metabolic type;

[0091] Depend on image 3 It can be seen that only the C line has exponential growth, but the A line has no exponential growth, indicating that the test result is a mutation homozygous CC, indica...

Embodiment 2

[0093] The detection result of embodiment two is:

[0094] Such as Figure 4 , Figure 5 and Figure 6 As shown, line A in the figure indicates that CYP2C9*3 is not detected, and line C indicates that CYP2C9*3 is detected.

[0095] Depend on Figure 4 It can be seen that only line A has exponential growth, but line C has no exponential growth, so the test result is wild homozygous AA;

[0096] Depend on Figure 5 It can be seen that both line A and line C have exponential growth, indicating that the test result is mutant heterozygous AC;

[0097] Depend on Figure 6 It can be seen that only line C has exponential growth, but line A has no exponential growth, indicating that the test result is mutation homozygous CC;

[0098] Thus, from Figure 4 , Figure 5 and Figure 6 The genotype of CYP2C9*3 was obtained in the analysis.

[0099] The detection result of embodiment three is:

[0100] Such as Figure 7 , Figure 8 and Figure 9 Shown:

[0101] Depend on Figur...

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Abstract

The invention discloses a kit for rapidly detecting CYP2C9*3 gene polymorphism. The kit comprises a PCR (Polymerase Chain Reaction) reaction mixing solution which comprises the following raw materials by final concentration: 0.05-0.12 U / microliter of DNA polymerase, 0.2 mM of dNTPs, 1 X of 5 X PCR buffering solution, 1.5-3.5 mM of MgCl2, 0.0005-0.015% (w / v) of lauryl sodium sulfate, 0.001-0.03% (w / v) of polyethylene glycol octylphenol ether, 0.5-1 micron of a forward primer, 0.5-1 micron of a reverse primer, 0.5-1 micron of a mutant molecular beacon, and 0.5-1 micron of a wild type molecular beacon; and the PCR reaction mixing solution is used for detecting cell samples. The primer, a Taqman-MGB probe and the kit for rapidly detecting the CYP2C9*3 gene polymorphism and the detection method of the CYP2C9*3 gene polymorphism are simple to operate, free from pollution, and rapid to detect.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer, a molecular beacon, a kit and a detection method for rapid detection of CYP2C9*3 gene polymorphism. Background technique [0002] Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP): refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. SNPs are associated with many diseases and determine the susceptibility of human diseases and the variability of drug responses. [0003] Molecular Beacon: It is a stem-loop double-labeled oligonucleotide probe that forms a hairpin structure of about 5-8 bases at the 5' and 3' ends. The nucleic acid sequences at both ends are complementary paired, and the fluorescent group labeled at one end is in close proximity to the quencher group labeled at the other end. Under this structure, the photons generated after the fluorophore is excited are quenched by the quencher, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2545/114C12Q2563/107
Inventor 贺庭祯罗德朋向·霄熊伟
Owner 重庆京因生物科技有限责任公司
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