A primer set, kit and method for hla gene amplification and genotyping

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

Active Publication Date: 2020-02-07
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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  • A primer set, kit and method for hla gene amplification and genotyping
  • A primer set, kit and method for hla gene amplification and genotyping
  • A primer set, kit and method for hla gene amplification and genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 Amplification of HLA gene

[0115] 1. Sample DNA extraction

[0116] The QlAamp blood extraction kit (QIAGEN) was used to extract DNA from 96 blood samples with known HLA genotypes. Use an ultraviolet spectrophotometer to measure the concentration of the extracted DNA sample, and adjust the concentration of the extracted DNA sample to 20-50ng / μl.

[0117] 2. Design HLA gene amplification primers

[0118] According to the latest HLA gene sequence in IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), such as Figure 1-Figure 3 As shown, the HLA-A and HLA-B genes have 8 exons, and the HLA-DRB1 gene has 6 exons. Find multiple groups (two per group) of suitable conserved regions, and ensure that each group of conserved regions can Cover the target area (HLA-A and HLA-B gene exons 1-8, HLA-DRB1 exons 2-3). Within the multiple groups of conserved regions found, design appropriate amplification primers respectively. If there are no conservative regions to choose from, use...

Embodiment 2

[0143] Example 2 Amplification of HLA 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 with an increase of less than or equal to 8 nucleotides at 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 example, 5'→3' at the 5'end increases GGCTACAT, and the 3'end 5'→3' increases TTTGAACC. The first set of amplification primers can be used to amplify Exons 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 with an increase of less than or equal to 8 nucleotides. In the example, a sequence of 1 nucleotide was added at the 5'end and 3'end of B-F2 and B-R2, respectively. In this example, 5'→3' at the 5'end increased G, and 5 at the 3'end '→3' increas...

Embodiment 3

[0145] Example 3 Amplification of 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 with an increase of less than or equal to 8 nucleotides at the 5'end and / or 3'end of B-F1 and B-R1. In this embodiment, one nucleotide is added to 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 example, C is added at the 5'end and G is added at the 3'end. The first set of amplification primers can amplify exons 1-3 of the HLA-B gene , The second set of amplification primers are sequences with an increase of less than or equal to 8 nucleotides at the 5'end and / or 3'end of B-F2 and B-R2. In this embodiment, they are in B-F2 and B-F2 and B-R2 respectively. -R2 has an 8 nucleotide sequence added to the 5'end and 3'end. In this example, 5'→3' of the 5'end adds CACAGTGT, and the 3'end 5'→3' adds CCCA...

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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, and specifically relates to a primer set, kit and method for HLA gene amplification and genotyping based on second-generation sequencing technology. Background technique [0002] Human leukocyte antigen (HLA), the coding gene is located on the short arm of chromosome 6, with a total length of about 4000Kb, 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 transplantation antigen, is an important factor in determining the level of transplant rejection. In organ transplantation, the higher the degree of 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 rejection is reaction. Therefore, efficient and accurate typing of HLA...

Claims

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Application Information

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