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Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination

A kit and site polymorphism technology, applied in the field of life science and biology, can solve the problems of reduced activity, low detection sensitivity, increase of oxygen free radicals, etc., and achieve the effect of high accuracy and good specificity

Inactive Publication Date: 2014-03-26
WUHAN ADICON CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the early 1990s, a study found that there was a C / T single nucleotide polymorphism (SNP) at the 609 site of NQO1 cDNA, which may change the secondary structure of the enzyme, reduce the activity of the enzyme, and increase the generation of oxygen free radicals
This technology is the first generation of DNA molecular marker technology, which uses restriction endonucleases to recognize specific sequences of DNA molecules and cuts DNA molecules at specific sequences to produce characteristic restriction fragments. The defect of this method is the detection The sensitivity is low and can only detect the approximate type, not the specific mutation ratio

Method used

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  • Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination
  • Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination
  • Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Primers and kits for detecting polymorphisms at the gene NQO1 site

[0056] Primers for polymorphism detection of quinone oxidoreductase 1 gene NQO1*2(C609T), NQO1*3(C465T), including amplification of quinone oxidoreductase 1 gene NQO1*2(C609T), Specific primers for NQO1*3 (C465T) site fragments and primers for pyrosequencing the obtained nucleic acid fragments, wherein,

[0057] The specific primer sequences for amplifying quinone oxidoreductase 1 gene NQO1*2 (C609T), NQO1*3 (C465T) site fragments from sample nucleic acid are:

[0058] C465T-F: 5'-CTAGCTTTACTCGGACCCACTC-3' (SEQ ID No. 1)

[0059] C465T-R: 5'-GCAACAAGAGGGAAGCTCCATC-3' (SEQ ID No. 2)

[0060] C609T-F:5'-TCTTACTGAGAAGCCCAGACCAACT-3' (SEQ ID No.3)

[0061] C609T-R: 5'-CTCCAGGCGTTTCTTCCATCC-3' (SEQ ID No. 4)

[0062] Among them, C465T-F and C609T-R are 5' biotin-labeled primers;

[0063] The primer sequences for pyrosequencing the obtained nucleic acid fragments are:

[0064] C465T-S: 5'-GC...

Embodiment 2

[0079] Example 2: DNA Extraction

[0080] DNA extraction reagents: red blood cell lysate, blood genomic DNA extraction kit (TIANGEN company).

[0081] Operation steps: take 500ul whole blood, put it into a 1.5ml centrifuge tube, add 1ml red blood cell lysate. Turn it up and down to mix completely, rotate and shake for 15 seconds, then put it into a centrifuge for centrifugation at 5000rpm for 10min. Pour off the upper layer, and it can be seen that there is a bloody precipitate at the bottom of the centrifuge tube. Add 500ul red blood cell lysate, repeat this lysis step once. Centrifuge at 5000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed. Add 20 μl proteinase K solution and mix well. Add 200 μl buffer GB, mix thoroughly by inverting, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap. Add 200 μl of a...

Embodiment 3

[0082] Embodiment 3: PCR amplification

[0083] The DNA extracted in Example 2 was amplified by conventional PCR using a PCR amplification system to obtain amplified DNA fragment amplification products.

[0084] The PCR amplification system used to amplify the NQO1*2 (C609T) site fragment of the quinone oxidoreductase 1 gene was 2*Buffer 10 μl, dNTP 4 μl, KOD enzyme 0.4 μl, upstream primer C609T-F 1 μl, downstream primer C609T-R 1 μl, pure Water 2.6 μl, add 1 μl template DNA to 19 μl amplification system, the total volume is 20 μl.

[0085]The PCR amplification system used to amplify the NQO1*3 (C465T) site fragment of the quinone oxidoreductase 1 gene was 2*Buffer 10 μl, dNTP 4 μl, KOD enzyme 0.4 μl, upstream primer C465T-F 1 μl, downstream primer C465T-R 1 μl, pure Water 2.6 μl, add 1 μl template DNA to 19 μl amplification system, the total volume is 20 μl.

[0086] Conventional PCR amplification reaction conditions are 94°C for 2 minutes; 98°C for 10 seconds, 60°C for 20 ...

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Abstract

The invention discloses a primer and a kit for detecting NQO1 gene polymorphism, and an application of primer and kit on pathological examination. The primer comprises a specific primer for amplifying the fourth exon C465T site fragment and the sixth exon C609T site fragment of quinone oxidoreductase 1 gene and a primer for pyrosequencing the obtained nucleic acid fragment. The polymorphism of the fourth exon C465T site fragment and the sixth exon C609T site fragment of quinone oxidoreductase 1 gene related to tumor susceptibility can be detected by adopting the pyrosequencing technology through the primer, method and kit disclosed by the invention, the specificity is good and the accuracy is high.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to primers, kits for detecting polymorphisms of quinone oxidoreductase 1 genes NQO1*2 (C609T), NQO1*3 (C465T), and pathological detection Application, using pyrosequencing technology, can detect the polymorphism of quinone oxidoreductase 1 gene, with good specificity and high accuracy. Background technique [0002] The quinone oxidoreductase 1 gene (NADPH: quinone oxidore-ductase1, NQO1) was first reported by Ernster and Navazio in 1958 and was originally called DT-lipoamide dehydrogenase (DT-diaphorase). It is a flavoprotease ubiquitous in most eukaryotic cells, which can detoxify many natural and synthetic compounds, and at the same time activate some quinone antineoplastic drugs, so it has attracted widespread attention. NQO1 is an inducible reductase, and external environmental factors such as phenolic antioxidants, polycyclic aromatic hydrocarbons, azo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6869C12Q2600/156
Inventor 钟丹薛群陈奕磊
Owner WUHAN ADICON CLINICAL LAB
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