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Micro-array chip with forty-five gene locus for detecting mitochondria diabetes

A microarray chip, mitochondrial gene technology, which is used in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc. The process takes a long time to achieve the effect of saving experimental time

Inactive Publication Date: 2007-08-15
WUHAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

The chip was used to detect known mutation samples screened by PCR-RFLP method, and the experimental conditions were initially explored. The disadvantages were that the chip did not set up a negative control, the background of Cy3 fluorescent dye was high, and the detected sites were few. (Liu Songmei, Zhou Xin, Li Xia, etc. Study on 8 mutation sites of mitochondrial gene in type 2 diabetes. Chinese Journal of Pathophysiology, 2006, 22: 586-591.)
The disadvantages are that the sample points of the membrane chip are not regular enough, the required washing and color development process takes a long time, there is a certain false positive rate and false negative rate in the visual observation and judgment results, and the entire mitochondrial mutation site cannot be systematically detected. Point (Liu Songmei, Zhou Xin, Zheng Fang, etc. Gene chip screening for mitochondrial DNA base mutations in type 2 diabetes. Chinese Journal of Medicine, 2006, 86(40): 2853-2857.)

Method used

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  • Micro-array chip with forty-five gene locus for detecting mitochondria diabetes
  • Micro-array chip with forty-five gene locus for detecting mitochondria diabetes
  • Micro-array chip with forty-five gene locus for detecting mitochondria diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Sample Handling and Labeling

[0054] (1) Fresh whole blood samples

[0055] The modified NaI method (Yu Hong, Peng Fangfang. Experimental Techniques of Medical Biochemistry and Molecular Biology, 1st edition, Wuhan University Press, 2003) was used to extract DNA templates.

[0056] (2) blood stain specimen

[0057] Take 1.5×1.5cm 2 For large or small blood stains, soak in 1ml of double-distilled water, shake and mix well, centrifuge at 12,000rpm for 3min, remove the supernatant, wash once with double-distilled water, then add 200μl of 5% Chelex-100, incubate at 56°C for 20-30min, Boil for 8 minutes, centrifuge at 10,000 rpm for 3 minutes, and take the supernatant for PCR amplification.

[0058] (3) hairball specimen

[0059] Cut off 3 to 4 hairs at a distance of 3 to 4 mm from the root, soak the hair ball in Chelex-100 solution, and keep it warm at 56°C for 6 to 10 hours. Oscillate for 5-10s. After boiling at 100°C for 10 minutes, centrifuge at 10,000 r / min for 3...

Embodiment 2

[0062] chip fabrication method

[0063] Dilute 90 amino-modified wild-type and mutant probes at 45 sites with 1×TE-Buffer (1mM Tris-HCl, 1mM EDTA, pH8.0) to a final concentration of 5 μM; The spotting matrix shown was arranged in a 384-well plate (Costar), and was spotted on the surface of an aldehyde-modified glass slide by a chip spotter (Nanoliter Dispensing system, Packard Instrument Company), and kept above a closed saturated saline solution. Relative humidity, fixed at room temperature (25°C-30°C) for 48-72 hours, stored in a 4°C refrigerator for later use.

Embodiment 3

[0065] Hybridization reactions and signal detection

[0066] Preheat the hybridization solution (Roche) at 42°C, fill it into the chip reaction chamber, and pre-hybridize for 30 minutes to block the non-specific binding of the substrate; take 45 μl of the denatured labeled PCR product mixture and mix it with the preheated hybridization solution at a ratio of 1:10, avoiding The reaction chamber was filled with light, and hybridized at 42°C for 3 hours.

[0067] Wash at room temperature (25°C-30°C) with 2×SSC-0.1% SDS for 5min×2 times (20×SSC-Buffer: 8.766g sodium chloride, 4.412g sodium citrate, distilled water to 50ml, hydrogenated Adjust the pH to 7.5 with sodium; 0.1% SDS: 1 gram of SDS (sodium dodecylsulfonate) was dissolved in 100ml of double distilled water), washed with 0.5×SSC-0.1%SDS at 42°C for 15min×2 times, double-distilled at room temperature Wash with distilled water for 10 minutes, and spin dry by centrifugation. The AxonGenepix 4000B chip scanner collected chi...

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Abstract

The invention discloses a micro array chip to check 45 gene locus of mitochondria diabetes, which comprises the following steps: choosing aldehyde slide as carrier; designing primer and probe; distributing and controlling chip dot matrix; distributing and controlling dot matrix of monitoring system and testing system; composing the monitoring system with negative contrast and parallel contrast of base point of discontinuity; setting the testing system as wild-type and saltant probe of mtDNA 45 site; setting each chip incorporates 1-3 0.04cm2 sampling point matrix, 90 trips probe of each matrix and 324 sites. The operation is simple, which can be used to check diabetes patient and high risk group.

Description

technical field [0001] The invention relates to a microarray chip with 45 gene sites for detecting mitochondrial diabetes. It can meet the requirements of hospitals at different levels, and is suitable for the detection of diabetic patients and high-risk groups. It is helpful for the correct diagnosis and classification of clinical special types of diabetes, early detection of patients, early prevention, and early treatment. Background technique [0002] Diabetes is a complex metabolic disease caused by defects in insulin secretion and / or insulin action. younger. Chronic complications caused by persistent hyperglycemia have become the main cause of renal failure, blindness and cardiovascular and cerebrovascular diseases, bringing a heavy burden to individual, social and national health care, and becoming a global social health and economic problem. Mitochondrial gene defects confer a certain genetic susceptibility to diabetes in individuals. In 199...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 刘松梅周新李霞郑芳涂建成汤冬玲曲喜英田艳丽蔡春林
Owner WUHAN UNIV
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