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Preparation and use of micro-array chip for HLA-B27 genotyping

An HLA-B27, microarray chip technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as excessive primer detection, error, and detection throughput is too small

Active Publication Date: 2009-01-28
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, these methods also have some technical problems, such as detection errors caused by too many primers, detection throughput is too small, etc.

Method used

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  • Preparation and use of micro-array chip for HLA-B27 genotyping
  • Preparation and use of micro-array chip for HLA-B27 genotyping
  • Preparation and use of micro-array chip for HLA-B27 genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Method 1: Design and Synthesis of HLA-B27 Genotyping DNA Microarray PCR Primers and Oligonucleotide Probes

[0031] In order to amplify the second exon of the HLA-B27 gene locus by specific PCR, design specific 5' primers PB1 and The 3' primer PB2 was designed at the sub-end position. See Table 2 for primer sequences.

[0032] Aiming at the polymorphism of the second exon gene sequence of HLA-B27, 19 types of typing probes were designed, as shown in Table 1.

[0033] Method 2: Preparation of HLA-DRB1 Genotyping DNA Microarray

[0034] 19 types of typing probes and two kinds of control probes were spotted on a specific area of ​​the glass slide by using a gene chip automatic spotting instrument to make a DNA microarray.

[0035] (1) Spotting probe solution: dilute the probe into 5×SSC, 0.05% SDS solution, the final concentration is 30uM;

[0036] (2) sampling matrix: (such as figure 1 ) spotting the probes on the glass slide, each probe was placed in the hybridization...

Embodiment 2

[0037] Example 2: Using HLA-B27 Genotyping to Detect Sample HLA-B27 Genotype with DNA Microarray

[0038] Take 10 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'.

[0039] Take a microarray slide, add 2ul of each of the 10 PCR products to the 10 wells of the chip, then add 8ul of hybridization solution (5XSSC) to each well, put the chip in the tank of the automatic hybridization washer, and set the hybridization temperature 52°C, hybridization time 40 minutes. Automatic washing and air drying after hybridization.

[0040] GnenPix4100A scanner is used to scan the hybridization signal to obtain the scanning image of the hybridization result, and convert the image into data.

[0041] Using analysis software, the above data is automatically analyzed and converted into the genotype of each sample, and the...

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Abstract

The invention relates to a gene detection kit used for clinical detection, in particular to the preparation and the usage of a micro-array chip used for HLA-B27 genotyping. A DNA micro-array chip technology is adopted, and the kit can carry out partign to human leucocyte antigen B27, with high pass, high efficiency and high specificity.

Description

Technical field [0001] The invention relates to a gene detection kit for clinical detection, in particular to a DNA microarray chip. The kit can type human leukocyte antigen B27 gene with high throughput, high efficiency and high specificity. 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 system of hum...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 胡守旺张帆王敏李明程钢何蕴韶
Owner GUANGZHOU DARUI BIOTECH
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