Primer group, kit and method for HLA (human leukocyte antigen) gene amplification and gene parting

A technology of gene amplification and typing method, which is applied in the field of gene detection and can solve the problems of inability to detect alleles and low resolution

Active Publication Date: 2018-08-24
BEIJING NUOSHI KANGYING MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this method is that it cannot detect new alleles, the resolution is not high, and the detection signal is an analog signal.

Method used

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  • Primer group, kit and method for HLA (human leukocyte antigen) gene amplification and gene parting
  • Primer group, kit and method for HLA (human leukocyte antigen) gene amplification and gene parting
  • Primer group, kit and method for HLA (human leukocyte antigen) gene amplification and gene parting

Examples

Experimental program
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Embodiment 1

[0114] The amplification of embodiment 1 HLA gene

[0115] 1. Sample DNA extraction

[0116] DNA was extracted from 96 blood samples of known HLA genotypes using the QlAamp blood extraction kit (QIAGEN). The concentration of the extracted DNA sample was measured by an ultraviolet spectrophotometer, and the concentration of the extracted DNA sample was adjusted to 20-50ng / μl.

[0117] 2. Design primers for HLA gene amplification

[0118] According to the latest HLA gene sequence in the IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), such as Figure 1-Figure 3As shown, the HLA-A and HLA-B genes have 8 exons, and the HLA-DRB1 gene has 6 exons. Find multiple groups (two for each group) of suitable conserved regions, and ensure that each group of conserved regions can Covers target region (HLA-A and HLA-B gene exons 1-8, HLA-DRB1 exons 2-3). Appropriate amplification primers were designed in the multiple groups of conserved regions found. If there is no conserved region to...

Embodiment 2

[0143] The amplification of embodiment 2HLA gene

[0144] This example is basically the same as Example 1, the only difference is that the first set of amplification primers is a sequence of less than or equal to 8 nucleotides added to the 5' end and / or 3' end of B-F1 and B-R1. In this embodiment, 8 nucleotides are added to the 5' end and 3' end of B-F1 and B-R1 respectively, and the nucleotides can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine), in this embodiment, 5'→3' at the 5' end increases GGCTACAT, and 5'→3' at the 3' end increases TTTGAACC, and the first set of amplification primers can be used to amplify Exon 1-3 of the HLA-B gene, the second set of amplification primers is the 5' end and / or 3' end of B-F2 and B-R2 to increase the sequence of less than or equal to 8 nucleotides, in this In the example, one nucleotide sequence is added at the 5' end and 3' end of B-F2 and B-R2 respectively. In this embodiment, 5'→3' at the 5' end increases G, a...

Embodiment 3

[0145] The amplification of embodiment 3 HLA gene

[0146] This example is basically the same as Example 1, the only difference is that the first set of amplification primers is a sequence of less than or equal to 8 nucleotides added to the 5' end and / or 3' end of B-F1 and B-R1. In this embodiment, one nucleotide is added at the 5' end and 3' end of B-F1 and B-R1 respectively, and the nucleotide can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine), in this embodiment, C is added at the 5' end, and G is added at the 3' end, and exons 1-3 of the HLA-B gene can be amplified using the first set of amplification primers , the second set of amplification primers is the 5' end and / or 3' end of B-F2 and B-R2 to increase the sequence of less than or equal to 8 nucleotides, in this embodiment, respectively in B-F2 and B-R2 -The 5' and 3' ends of R2 increase the sequence of 8 nucleotides. In this embodiment, the 5'→3' at the 5' end increases CACAGTGT, and the 5'→3'...

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Abstract

The invention provides a primer group, a kit and a method for HLA (human leukocyte antigen) gene amplification and gene parting and belongs to the field of gene detection. According to the primer group for HLA gene amplification, primers are designed according to 8 exon zones of an HLA-A gene, 8 exon zones of an HLA-B gene and an exon 2 and an exon 3 of an HLA-DRB1 gene, PCR amplification is performed on the HLA gene by use of the primers, and a product with a gene segment length larger than 400 bp and smaller than 1.5 kb is obtained. By means of the PCR amplified HLA genes, the gene segment length of the amplification product is larger than 400 bp and smaller than 1.5 kb, the requirement for completeness of a template is reduced, the amplification efficiency is high, the amplification reaction time is short, the cost is reduced, and the demands of large-scale amplification or gene parting of sample HLA genes can be met.

Description

technical field [0001] The invention belongs to the field of gene detection, in particular to a primer set, kit and method for HLA gene amplification and genotyping based on next-generation sequencing technology. Background technique [0002] Human leukocyte antigen (human leukocyte antigen, referred to as HLA), the encoding gene is located on the short arm of chromosome 6, with a total length of about 4000Kb. It is the most complex genetic polymorphism system known to humans and is closely related to the function of the human immune system. HLA, also known as transplant antigen, is an important factor in determining the level of transplant rejection. During organ transplantation, the higher the HLA compatibility between the donor and the recipient, the lower the incidence of rejection, the higher the success rate of transplantation and the long-term survival rate of transplanted organ patients; on the contrary, the more likely to occur rejection reaction. Therefore, effic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2535/122
Inventor 康颖
Owner BEIJING NUOSHI KANGYING MEDICAL TECH
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