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Chip for detecting human DNA repair enzyme genetic polymorphism and preparation thereof

A gene chip and repair enzyme technology, applied in the field of human DNA repair enzyme gene polymorphism chip and its preparation

Inactive Publication Date: 2012-07-18
SHANDONG TUMOR HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chip can overcome the defect that the existing technology cannot detect multiple gene polymorphisms at one time, and meet the needs of disease prevention and control and clinical medical fields

Method used

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  • Chip for detecting human DNA repair enzyme genetic polymorphism and preparation thereof
  • Chip for detecting human DNA repair enzyme genetic polymorphism and preparation thereof
  • Chip for detecting human DNA repair enzyme genetic polymorphism and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] (1) Glass slide treatment: take a conventional bare glass sheet without chemical modification and soak it in an 8% glutaraldehyde aqueous solution for 1 hour to obtain an aldylated glass sheet as the substrate of the chip.

[0105] (2) Determining the probes: According to the gene sequence of the polymorphic site of the DNA repair enzyme gene, select and determine 6 specific detection probes, the gene sequences of which are respectively:

[0106] (XRCC1-194)-w: 5'-(NH2)-(CH2)6-Pol(dT)15-AAGAAGTCGGCCTAGTTG;

[0107] (XRCC1-194)-m: 5'-(NH2)-(CH2)6-Pol(dT)15-AAGAAGTCGAGGTAGTTG;

[0108] (XRCC1-399)-w: 5'-(NH2)-(CH2)6-Pol(dT)15-ACGGGAGGGTCTCCATT;

[0109] (XRCC1-399)-m: 5'-(NH2)-(CH2)6-Pol(dT)15-ACGGGAGGGCCTCCATT;

[0110] (XPD-312)-w: 5'-(NH2)-(CH2)6-Pol(dT)15-CACGACGGGTTGCTTCA;

[0111] (XPD-312)-m: 5'-(NH2)-(CH2)6-Pol(dT)15-CACGACGGGCTGCTTCA;

[0112] Choose to determine 1 positive control probe whose gene sequence is:

[0113] β-actin: 5'-(NH2)-(CH2)6-Pol(dT)15-TAC...

Embodiment 2

[0120] (1) Use Omega Whole Blood DNA Extraction Kit to extract genomic DNA from peripheral blood samples according to the instructions, and dissolve DNA with sterilized ultrapure water.

[0121] (2) PCR amplification of gene fragments containing each site

[0122] Design specific upstream and downstream primers for XRCC1Arg194Trp, Arg399Gln and XPDAsp312Asn polymorphic sites, specifically:

[0123] XRCC1Arg194Trp site: upstream primer 5'-AGCAGCCCACCTATAATACTG-3'

[0124] Downstream primer 5'-Tamara-CTCAGACCCAGGAATCTGAG-3'

[0125] XRCC1Arg399Gln site: upstream primer 5'-CCCTCAGATC ACACCTAACTG-3'

[0126] Downstream primer 5'-Tamara-AGTAGTCTGCTGGCTCTGGGC-3'

[0127] XPDAsp312Asn site: upstream primer 5'-AGGATCAAAGAGACAGACGAG-3'

[0128] Downstream primer 5'-Tamara-TCTGCGAGGAGACGCTATCAG-3'

[0129] Positive control gene β-actin: upstream primer 5'-TTAGTTGCGTTACACCCTTTC-3'

[0130] Downstream primer 5'-Tamara-GAACGGTGAAGGTGACAGC-3'

[0131] The above downstream primers were ...

Embodiment 3

[0137] (1) Use Omega Whole Blood DNA Extraction Kit to extract genomic DNA from peripheral blood samples according to the instructions, and dissolve DNA with sterilized ultrapure water.

[0138] (2) PCR amplification of gene fragments and β-actin gene fragments including each site. The above downstream primers were labeled with Tamara fluorescence, and the PCR amplification reaction of the target gene was carried out. The PCR amplification reaction system is: 20 μl PCR reaction system, the mixture contains 5 μl template DNA, 0.4 μl each of 20 mM upstream and downstream primers, 0.4 μl 10 mM dNTP, 25 mM MgCl 2 1.2μl, 5U / μl Taq polymerase 0.2μl and 10×PCR Buffer 2μl. Take 5 μl of the PCR product, and use 1% agarose gel electrophoresis containing ethidium bromide for electrophoresis to observe the results.

[0139] (3) Hybridization of PCR amplification product and human DNA repair enzyme gene detection chip: the target DNA amplification product labeled with Tamara fluorescence...

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Abstract

The invention discloses a glass gene chip for detecting human DNA repair enzyme genetic polymorphism, which comprises a glass substrate subjected to aldehyde treatment, and oligonucleotide probe, control and blank spot coatings distributed on the glass substrate in an array, wherein the locus coatings comprise six strips of different-locus DNA repair enzyme gene-specific detection probes, one strip of positive control, three strips of negative control and blank spot sample solution control. The detection probes and control probes of the glass gene chip all take poly(dT)15 as connecting arms, and amino-group-containing six-carbon groups are added into the 5' ends of the probes. The invention also discloses the design of an upstream primer and a downstream primer for the amplification of DNA samples in human whole blood. The chip of the invention can quickly and highly-efficiently perform simultaneous detection on the polymorphism of human DNA repair enzyme genes at XRCC1Arg194Trp locus, Arg399Gln locus and XPDAsp321Asn locus so as to know the capacity of organisms in repairing DNA damage caused by endogenic factors and exogenous factors, and has great significance for the judgment of risks that individuals suffer from malignant tumors, guidance for patients with the tumors to select proper chemotherapeutical medicaments and the execution of individualized therapy.

Description

technical field [0001] The invention relates to a human DNA repair enzyme gene polymorphism chip and its preparation, in particular to a glass gene chip capable of simultaneously detecting gene polymorphisms of XRCC1 Arg194Trp, Arg399Gln sites and XPD Asp312Asn sites and a preparation method thereof. Background technique [0002] Gene chip refers to the use of many specific oligonucleotide fragments or gene fragments as probes, which are regularly arranged and fixed on a solid support, and then hybridized with the labeled nucleic acid sample to be tested according to the principle of base pairing, and then The hybridization signal is detected by a certain detection system, and a computer system is used to analyze and process the data of each hybridization signal, so as to quickly obtain the desired information. Compared with the traditional hybridization technology, this technology has the remarkable characteristics of miniaturization, high sensitivity, high parallelism, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 刘杰宋宝王哲海白雪丽刘曙光张政伟郑燕吕丽燕
Owner SHANDONG TUMOR HOSPITAL
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