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

A technology of HLA-A and genotyping, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as primer detection errors, low detection throughput, and reduced specificity

Active Publication Date: 2011-02-09
DAAN GENE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, these methods also have some technical problems, such as detection errors caused by too many primers, specificity reduction caused by single temperature of probe hybridization, low detection throughput, etc.

Method used

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

Examples

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Embodiment 1

[0022] Method 1: Design and Synthesis of HLA-A Genotyping DNA Microarray PCR Primers and Oligonucleotide Probes

[0023] In order to use specific PCR to amplify the 2nd and 3rd exons of the HLA-A gene locus, respectively at the position from the end of the 1st intron to the beginning of the 2nd exon, and from the end of the 2nd intron to the 3rd intron Design specific 5'-end primers Pa1 and Pa2 at the beginning of the exon, and design 3'-end primers Pa3 and Pa4 at the end of the second exon and the end of the third exon. Synthesis, CY3 labeling, purification. See Table 2 for primer sequences.

[0024] Aiming at the polymorphism of exons 2 and 3 of HLA-A gene, 53 types of typing probes were designed, the sequences are shown in Table 1, synthesized, modified and purified.

[0025] Method 2: Preparation of HLA-A Genotyping DNA Microarray

[0026] A gene chip automatic spotting instrument was used to spot 53 types of typing probes and two kinds of control probes on a specific a...

Embodiment 2

[0030] Example 2: Using HLA-A genotyping to detect HLA-A genotypes in clinical samples using DNA microarrays Take 16 DNA samples of known genotypes, use CY3-labeled PCR primers, and the upstream and downstream primers are 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'.

[0031] 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.

[0032] GnenPix4100A scanner is used to scan the hybridization signal to obtain the scanning image of the hybridization result, and convert the image into ...

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Abstract

The invention relates to a gene test kit used for clinical detection, in particular to the preparation and the usage of a micro-array chip used for HLA-A genotyping. A DNA micro-array chip technology and a dual temperature hybridization method are adopted, and the kit can carry out parting to the HLA-A gene, 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 DNA microarray chip technology and dual temperature hybridization method, the kit can analyze HLA-A gene with high throughput, high efficiency and high specificity. type. 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 th...

Claims

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Application Information

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