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HLA-C gene typing DNA micro-array chip reagent kit

A microarray chip, HLA-C technology, applied in genetic engineering, plant genetic improvement, microbial measurement/inspection, etc., can solve problems such as low detection throughput, errors, excessive primer detection, etc.

Active Publication Date: 2011-03-23
DAAN GENE CO LTD +1
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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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  • HLA-C gene typing DNA micro-array chip reagent kit

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Embodiment 1

[0020] Method 1: Design and synthesis of HLA-C genotyping DNA microarray PCR primers and oligonucleotide probes Design specific primers P1 and P2 at the beginning and end of the exon, and design primers P3 and P4 at the beginning and end of the third exon. Synthesis, CY3 labeling, purification. See Table 2 for primer sequences.

[0021] Aiming at the polymorphism of exon 2 and exon 3 of HLA-C gene, 22 types of typing probes were designed, whose sequences are shown in Table 1, synthesized, modified and purified.

[0022] Method 2: Preparation of HLA-C Genotyping DNA Microarray

[0023] 22 kinds of typing probes and two kinds of control probes were spotted on the specific area of ​​the glass slide by using a gene chip automatic spotting instrument 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 ) spotting the probes on the glass s...

Embodiment 2

[0026] Example 2: Detection of HLA-C genotype in clinical samples using DNA microarray for HLA-C genotyping

[0027] 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'.

[0028] Take a microarray slide, add 2ul of each of the 10 kinds of PCR products to the 10 wells of the chip, then add 8ul of hybridization solution (5×SSC) to each well, put the chip into the tank of the automatic hybridization washer, set The hybridization temperature was 52° C., and the hybridization time was 40 minutes. Automatic washing and air drying after hybridization.

[0029] GnenPix4100A scanner is used to scan the hybridization signal to obtain the scanning image of the hybridization result, and convert the image into data.

[0030] Using analysis software, the above data is automatically analyzed and converted into ...

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Abstract

The invention relates to a gene detecting kit that is used in clinical detection, in particular to a DNA microarray chip kit, which can realize subtype detection of human leukocyte antigen C(HLA-C) gene in high flux, 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 kit, which can perform typing detection on HLA-C gene with high throughput, high efficiency and high specificity. Background technique [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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/12
Inventor 胡守旺张帆李明程钢何蕴韶
Owner DAAN GENE CO LTD
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