HLA-DPA1 gene typing kit

A technology of AMP-DPA1-F3 and genotyping, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of increasing the risk of disease, achieve improved matching effect, simple steps, The effect of improving accuracy

Pending Publication Date: 2020-09-15
银丰基因科技有限公司 +1
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AI Technical Summary

Problems solved by technology

In chronic hepatitis B, HLA-DPA1*02:02-DPB1*05:01 and HLA-DPA1*02:02-DPB1*03:01 in HLA-DPA1 and -DPB1 genes in Japanese and Thai populations were found to be Susceptible type of chronic hepatitis B, while HLA-DPA1*01:03-DPB1*04:02 and HLA-DPA1*01:03-DPB1*04:01 are protective haplotypes, persistent chronic hepati...

Method used

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  • HLA-DPA1 gene typing kit
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  • HLA-DPA1 gene typing kit

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Experimental program
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Embodiment 1

[0032] The design of embodiment 1 primer

[0033] The primer design software uses primer premier 5. The designed primers are based on the consecutive bases including polymorphic sites in the HLA-DPA1 locus gene sequence in the IMGT / HLA database (https: / / www.ebi.ac.uk / ipd / imgt / hla / ) obtained from the base sequence. All primers were designed to avoid currently known mutation sites or use degenerate base methods to avoid typing errors due to missed detection of sites. In the present invention, 3 forward amplification primers and 2 reverse amplification primers have been designed from the beginning to the final completion, and 6 pairs of amplification primers have been combined to design the 4 exons of 1-4 exons respectively. A forward sequencing primer and reverse sequencing primer, the amplification primer sequence is as shown in Table 1 (as shown in SEQ ID NO.1~5), and the sequencing primer is as shown in Table 2 (as shown in SEQ ID NO.6~13) Show).

[0034] Utilize above-me...

Embodiment 2

[0040] The detection of embodiment 2 HLA-DPA1 genotyping

[0041] The specific implementation steps are as follows:

[0042] 1. Genomic DNA extraction: Extract according to the operation manual of the automatic DNA extraction kit, use a microplate reader to measure the concentration of the extracted DNA sample, the purity should be between 1.8-2.0, otherwise re-extract DNA.

[0043] 2. HLA-DPA1 gene PCR amplification: the DNA concentration was adjusted to 30 ng / μl, and the reaction system during the amplification process was shown in Table 3.

[0044] table 3

[0045] Template DNA (30ng / μl) 2μl Forward primer (10pM) 1μl Reverse primer (10pM) 1μl dNTP 1.6μl 10x LA Taq buffer (Mg 2+ )

2μl enhancer 2μl 10x LA Taq 0.2μl wxya 2 o

10.2μl total capacity 20μl

[0046] The amplification procedure is as follows:

[0047] Denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 60°C for ...

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Abstract

The invention discloses an HLA-DPA1 gene typing kit. The HLA-DPA1 gene typing kit comprises two amplification primers and four sequencing primers shown in SEQ ID NO. 3, 4, 8, 9, 10 and 11. The kit canserve as an independent and widely-applied identification method, can successfully achieve accurate typing identification of HLA-DPA1 sites, can complete full-length amplification of HLA-DPA1 genes at a time, and then conducts double-end sequencing on 2 and 3 exons. Compared with the prior art, the HLA-DPA1 gene typing kit has the advantages that the steps are simple, the typing speed is high, typing is accurate, a report can be issued within three days, and the kit can be compatible with first-generation and second-generation sequencing platforms. The improvement of a typing method is beneficial for improving the accuracy of HLA matching of hematopoietic stem cell transplantation donors and recipients, therefore, more suitable transplantation donors are selected, the rejection reaction in the transplantation process is reduced, and evidence can be provided for clinicians to diagnose disease occurrence.

Description

technical field [0001] The invention relates to an HLA-DPA1 high-resolution genotyping kit, which belongs to the technical field of gene detection. Background technique [0002] The major histocompatibility complex (MHC) is the most complex genetic polymorphism system in humans, and the human MHC product is usually called HLA (human leukocyte antigen, HLA), that is, human leukocyte antigen. The MHC gene is located in the 6p21.31 region of the short arm of human chromosome 6 and consists of a series of closely linked loci with a total length of about 4000kb. According to the characteristics of HLA molecular structure and distribution, it is divided into three types of molecules: HLA-Ⅰ, HLA-Ⅱ and HLA-Ⅲ. The molecular functions are mainly related to the synthesis of part of the immune-related complement system and inflammation-related factors. [0003] Accurate HLA typing technology has been widely used in organ and hematopoietic stem cell transplantation to find HLA-matched ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2535/101C12Q2521/301C12Q2565/125C12Q2535/122
Inventor 王燕张倩刘克瑶王林林王连水杜昭励杨海燕
Owner 银丰基因科技有限公司
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