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Reagent kit for parting detection of HLA-DRB1 gene

A technology for HLA-DRB1 and genotyping, which is applied in the field of human leukocyte antigen gene detection and typing kits, can solve the problems of low probe hybridization specificity, improve specificity, increase detection throughput, and avoid Effect of false positive and false negative problems on probe hybridization results

Active Publication Date: 2010-12-29
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing DNA microarray methods all use single-temperature hybridization, which will reduce the hybridization specificity of some probes, which is an inherent shortcoming in the methodology.

Method used

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  • Reagent kit for parting detection of HLA-DRB1 gene
  • Reagent kit for parting detection of HLA-DRB1 gene
  • Reagent kit for parting detection of HLA-DRB1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Design and synthesis of HLA-DRB1 genotyping DNA microarray PCR primers and oligonucleotide probes

[0021] In order to amplify the second exon of the HLA-DRB1 gene locus by specific PCR, design specific 5' primers P1-1, P1-2 from the end of the first intron to the beginning of the second exon respectively , P1-3, P1-4, P1-5 and P1-6, and the 3'common primer P2 was designed at the end position of exon 2. (see Table 2 of the Summary of the Invention for the primer sequences) for the polymorphism of the 2nd exon gene sequence of HLA-DRB1, design 33 kinds of typing probes, the probes are divided into two groups (see Table 1 of the Summary of the Invention), the first A group of 24 probes, the hybridization temperature is 52°C; a second group of 9 probes, the hybridization temperature is 45°C.

Embodiment 2

[0022] Example 2: Preparation of HLA-DRB1 Genotyping DNA Microarray

[0023] Using a gene chip automatic spotting instrument, 33 types of typing probes and two kinds of control probes were spotted on a specific area of ​​the glass slide to make a DNA microarray.

[0024] (1) Spotting probe solution: dilute the probe into 5×SSC, 0.05% SDS solution, the final concentration is 30uM;

[0025] (2) sampling matrix: (such as figure 1 ) divides the probes into two groups, the first group contains 24 typing probes and 2 control probes. The first group of probes was spotted on the first glass slide and divided into 16 hybridization areas, and the hybridization areas were arranged in 2 columns and 8 rows. In each hybridization area, the probes are arranged in the following manner: 3 points for each probe, 3 probes in each row, 9 points, 9 rows in total, and only 2 controls in the 9th row, 6 points.

[0026] The second group contains 9 typing probes and 2 control probes. The arrangeme...

Embodiment 3

[0027] Example 3: Using HLA-DRB1 genotyping detection and result analysis

[0028] Take 16 DNA samples with known genotypes, use CY3-labeled PCR primers, upstream and downstream primers 1:30 (molecular ratio) asymmetric PCR method, PCR cycle parameters are: 94°C 5'; 94°C 30", 58°C 30", 72°C 30", 40 cycles; 72°C 5'.

[0029] Take a pair of microarray slides, add 2ul of each of the 16 PCR products to the 16 wells of the first slide and the 16 wells of the second slide, then add 8ul of hybridization solution (5XSSC) to each well, and place the slides in automatic hybridization In the two tanks of the washer, set the hybridization temperature: 52°C for the first piece and 45°C for the second piece. The hybridization time was 40 minutes. Automatic washing and air drying after hybridization.

[0030] GnenPix4100A scanner is used to scan the hybridization signal to obtain the scanning image of the hybridization result, and convert the image into data.

[0031] Using analysis soft...

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Abstract

The invention relates to a gene detecting kit used for clinical detection, in particular to the kit which adopts a DNA micro-array chip and a dual-temperature hybridization probe to execute the detection and typing on human leukocyte antigen gene in a high-throughput, high-efficiency and high-specificity manner and the usage thereof.

Description

Technical field [0001] The invention relates to a gene detection kit, in particular to a kit for detecting and typing the human leukocyte antigen gene by using DNA microarray and dual-temperature hybridization technology and its preparation and use method. technical background [0002] HLA (Human leukocyte antigen, human leukocyte antigen) is the main human histocompatibility system. Histocompatibility refers to the degree of acceptance by both the donor and the recipient when tissue or organ transplantation is performed between different individuals. The reaction caused by the incompatibility of donor and recipient tissue has been proved to be an immune response, which is induced by alloantigens on the cell surface. This antigen representing individual specificity is called transplantation antigen or histocompatibility antigen, which plays The main antigen is called the major histocompatibility system (MHS), and HLA (human leukocyte antigen) is the major histocompatibility...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 胡守旺张帆胥顺程钢何蕴韶
Owner DAAN GENE CO LTD
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