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Kit for rapidly detecting CYP2D6 gene copy number by adopting pyrosequencing method and applications of kit

A pyrosequencing method, a technology of gene copy number, which can be used in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., and can solve the problems of low reliability, high cost, and slow progress in individualized treatment. , to achieve the effect of easy operation, small sample size, and great promotion and application value

Inactive Publication Date: 2017-06-20
HANGZHOU D A GENETIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high cost and low reliability of the current detection methods for CYP2D6 gene copy number, many researchers have enough energy to spare. At the same time, due to economic reasons, the development of gene detection for individualized treatment is slow

Method used

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  • Kit for rapidly detecting CYP2D6 gene copy number by adopting pyrosequencing method and applications of kit
  • Kit for rapidly detecting CYP2D6 gene copy number by adopting pyrosequencing method and applications of kit
  • Kit for rapidly detecting CYP2D6 gene copy number by adopting pyrosequencing method and applications of kit

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Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Reagents.

[0042] (1) DNA extraction reagents:

[0043] Purchased from QIAGEN Company.

[0044] (2) Reaction solution:

[0045] PCR Buffer: purchased from Fermentas, USA;

[0046] Primers SEQ ID NO: 1-2, synthesized by Shanghai Yingjun Biotechnology Co., Ltd.;

[0047] MgCl 2 : purchased from the U.S. Fermentas company;

[0048] 0.2mM dNTPs: purchased from Fermentas, USA;

[0049] 2U / μL Taq DNA polymerase: purchased from Fermentas, USA.

[0050] (3) Reagent for single-strand purification:

[0051] 75% (v / v) ethanol solution: purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.;

[0052] 0.2mol / LNaOH: purchased from Shanghai Shisi Hewei Chemical Co., Ltd.;

[0053] 10mmol / L Tris-Acetate (pH 7.6): Tris-base was purchased from Sigma Company in the United States, and anhydrous acetic acid was purchased from Hangzhou Chemical Reagent Co., Ltd.;

[0054] Binding buffer: 10mM Tris-HCl (Tris-base was purchased from Sigma, USA; hydrochloric acid wa...

Embodiment 2

[0062] Embodiment 2: detection method.

[0063] Instruments: Bio-Rad S1000 PCR instrument, Beckman Microfuge 22R desktop micro-refrigerated centrifuge, QIAGEN PyroMark Q96ID sequencer.

[0064] (1) Extract DNA from whole blood samples:

[0065] Referring to the published literature, the corresponding commercial DNA extraction kit was used, and the genomic DNA in the blood was prepared according to the kit instructions, and used as a PCR reaction template for future use.

[0066] After the DNA is dissolved, use a Nano-Space ultraviolet spectrophotometer to measure the nucleic acid concentration. If the nucleic acid concentration is greater than 50ng / uL, it is considered qualified. Store the nucleic acid specimen in a 4°C refrigerator;

[0067] (2) using the DNA obtained in step (1) as a template, and using specific primers to perform PCR amplification;

[0068] Among them, PCR amplification adopts 50μl system, in which DNA template 2μL, 10×PCR buffer 5μL, MgCl 2 (25mmol / L) 4...

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Abstract

The invention discloses primers for detecting the CYP2D6 gene copy number. The primers comprise: (1) a primer pair for amplifying the CYP2D6 gene, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2, and the 5' terminal of the downstream primer is labeled by biotin; and (2) a sequencing primer for the CYP2D6 gene copy number, wherein the nucleotide sequence of the sequencing primer is shown as SEQ ID NO:3. The invention further discloses applications of the primers in detecting the CYP2D6 gene copy number. The detection method is high in sensitivity, the result is visual, the interpretation is simple, accurate and rapid, the accurate, rapid and high-throughput detection for the CYP2D6 copy number can be realized, and thus the primers have great popularization and application values in the personalized medical treatment.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a kit for rapidly detecting the copy number of CYP2D6 gene by pyrosequencing method and an application thereof. Background technique [0002] Human cytochrome P450s (cytochrome P450s, CYP) is a large family composed of many isoenzymes, mainly located in liver microsomes, involved in the biotransformation of endogenous and exogenous substances, and in regulating the interaction between the body and the external environment. It plays an important role in maintaining the stability of the internal environment of the body. At present, there are more than 30 kinds of P450 known in humans, and there are seven important ones, and CYP2D6 is one of them. [0003] CYP2D6, also known as isoquinidine 4-hydroxylase, was first purified from liver microsomes of fast metabolizers in 1984. It is mainly distributed in liver, small intestine and brain tissue, accounting for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/301C12Q2537/16
Inventor 任绪义施宏虞闰六
Owner HANGZHOU D A GENETIC ENG
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