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Testing chip in cytokine gene type and application

A gene chip and genotype technology, which is applied to the gene detection chip for detecting tumor necrosis factor, interleukin-6 genotype, and the field of detecting TNF and IL-6 genotype, can solve the problem of difficult control and inability to widely develop TNF and IL-6 genes. There are many factors in the type test and test results, etc.

Inactive Publication Date: 2005-08-31
JINAN CENTER HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current methods for determining TNF and IL-6 genotypes mainly use methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), manual or automatic sequencing, and sequence-specific primer PCR. The detection period is long, and there are many factors affecting the detection results, which are difficult to control and difficult to meet the requirements of practical application, so that the detection of TNF and IL-6 genotypes has not been widely carried out in drug research units and clinics.

Method used

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  • Testing chip in cytokine gene type and application
  • Testing chip in cytokine gene type and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: the preparation of gene chip

[0055] (1) Purchase aldehyde-modified glass slides (product number: BSM03011, Shanghai Bio Technology Co., Ltd.). The following probes were artificially synthesized (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), dissolved in water to a concentration of (100pmol / ul), and then mixed in equal proportions with 2× spotting buffer (product number: BST02010, Shanghai Bio-Technology Co., Ltd.) . Then, use the GMS417 sample spotting instrument of Affymetrix company to make points such as figure 1 array of . Then leave overnight at room temperature.

[0056] Wherein each probe sequence is:

[0057] TNF-α / -308G: NH 2 -5'-tttttttttttttttt-cccgtccCcatgcc-3' (14 poly)

[0058] (SEQ ID NO: 11)

[0059] TNF-α / -308A: NH 2 -5'-tttttttttttttttt-cccgtccTcatgccc-3' (15 poly)

[0060] (SEQ ID NO: 12)

[0061] TNF-α / -238G:NH 2 -5'-tttttttttttttttt-cctgctcCgattccga-3 (16 poly)

[0062] SEQ ID NO: 13)

[0063] TNF-α / -238A...

Embodiment 2

[0077] Example 2: Preparation of chromosomal DNA

[0078] Take 500 μl of whole blood from the subject to be tested, draw it with a disposable sterile injection needle, collect it in a sterile 1.5ml centrifuge tube containing 50ul of 2% EDTA solution, cover the tube cap, turn it upside down several times, and mix well. Take a sterile 1.5ml centrifuge tube, add 100μl of the whole blood to be tested and 300μl of pure water into it, mix well, and let stand at room temperature for 8 minutes; put the centrifuge tube in a centrifuge and centrifuge at 6000g for 1 minute; take out the centrifuge tube, pour off the supernatant, and a small amount of white precipitate can be seen at the bottom of the centrifuge tube; add 60 μl of extract solution (product number: BST01010, Shanghai Bio-Tech Co., Ltd.) ;Take out the centrifuge tube, put it in a centrifuge, and centrifuge at 12,000g for 15 minutes. The supernatant after centrifugation can be directly used for PCR amplification.

Embodiment 3

[0079] Embodiment 3: Amplify HLA-B gene fragment by PCR method

[0080] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize the following primers:

[0081] TNF upstream primer: Biotin-5'-tccctccaaccccgttttct-3'

[0082] TNF downstream primer: 5'-catctggaggaagcggtagtgg-3'

[0083] IL-6 upstream primer: 5'-cacactccacctggagacgcc-3'

[0084] IL-6 downstream primer: 5'-Biotin-agcgggtggggctgattgga 3'

[0085] reaction system

[0086] Use a PCR amplification instrument Tc-25 / H (Hangzhou Dahe Thermal Magnetic Electronics Co., Ltd.) to amplify according to the following procedure: 94°C for 5 minutes, then 40 cycles of 94°C for 25sec, 56°C for 25sec, and 72°C for 25sec, and finally 72°C. ℃ 5min.

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Abstract

A gene chip for detecting INF and IL-6 genotypes, a reagent kit containing said gene chip for detecting INF and IL-6 genotypes, and its application for predicting the information about the danger of the diseases associated with said genes and providing the method to develop the relative medicines are disclosed.

Description

(1) Technical field [0001] The invention relates to a gene analysis and detection product, specifically a gene detection chip, more specifically a gene detection chip suitable for detecting tumor necrosis factor (TNF) and interleukin 6 (IL-6) genotypes. The invention also relates to methods for detecting TNF and IL-6 genotypes. (2) Background technology [0002] Since the 1980s, with the rapid development of molecular biology, research on TNF and IL-6 has continued to deepen, and it has been found that it has a variety of biological activities. It can induce apoptosis (Apoptosis) on some non-tumor cells and most tumor cells. When the body is in a pathological state of inflammation such as virus, bacteria, parasite infection, or trauma, these functions play an important role in clearing damaged cells and maintaining the balance of the body's internal environment. However, TNF and IL-6 genotypes are different, and the secretion of tumor necrosis factor, interleukin 6 and oth...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 汪运山贾堂宏朱滨孙悦张雨
Owner JINAN CENTER HOSPITAL
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