A primer set, kit and method for high-resolution typing of hla-drb1 gene

An HLA-DRB1, high-resolution technology, applied in the field of primer sets for HLA-DRB1 genotyping, can solve problems such as difficult amplification, high requirements for DNA template integrity, and inability to meet HLA-DRB1 typing

Active Publication Date: 2019-09-13
BEIJING NUOSHI KANGYING MEDICAL TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Therefore, the technical problem to be solved by the present invention is that the existing HLA-DRB1 gene full-length fragment is relatively long, the DNA template integrity requirement is high, the amplification is difficult, the cost is high, and it cannot meet the problem of large-scale sample HLA-DRB1 typing. The invention provides a primer set, kit and method for HLA-DRB1 gene amplification and high-resolution typing

Method used

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  • A primer set, kit and method for high-resolution typing of hla-drb1 gene
  • A primer set, kit and method for high-resolution typing of hla-drb1 gene
  • A primer set, kit and method for high-resolution typing of hla-drb1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1 Amplification of HLA-DRB1 gene

[0096] 1. Sample DNA extraction

[0097] DNA was extracted from 96 blood samples with known HLA-DRB1 genotypes using the QlAamp Blood Extraction Kit (QIAGEN). The concentration of the extracted DNA sample is determined by using an ultraviolet spectrophotometer, and the concentration of the extracted DNA sample is adjusted to 20-50 ng / μl.

[0098] 2. Design primers for HLA-DRB1 gene amplification

[0099] According to the latest HLA-DRB1 gene sequence in the IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), such as figure 1 As shown, the HLA-DRB1 gene has 6 exons, multiple groups (two for each group) of suitable conserved regions are searched, and each group of conserved regions is guaranteed to cover exons 2-3 of the HLA-DRB1 gene. In the found multiple groups of conserved regions, design appropriate amplification primers respectively. When several allele sequences are inconsistent, use degenerate bases, or design a new...

Embodiment 2

[0112] Example 2 Amplification of HLA-DRB1 Gene

[0113] This example is basically the same as Example 1, the only difference is that the 5' end and / or 3' end of the first group of amplification primers has a sequence of less than or equal to 8 nucleotides, in this example, respectively in DRB1 -F2-1, DRB1-F2-2, DRB1-F2-3, DRB1-F2-4, DRB1-F2-5, DRB1-F2-6, DRB1-F2-7 and DRB1-2R 5' end and 3 8 nucleotides are added to the 'end, and the nucleotides can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine). In this embodiment, the 5' end 5'→3' increases GGCTACAG, 5'→3' at the 3' end increases TTTGAACC, the exon 2 of the HLA-DRB1 gene can be amplified using the first set of amplification primers, and the 5' end of the second set of amplification primers And / or a sequence with an increase of less than or equal to 8 nucleotides at the 3' end, in this embodiment, a sequence with 1 nucleotide increase at the 5' end and 3' end of DRB1-F3 and DRB1-R3, respectively, In ...

Embodiment 3

[0114] Example 3 Amplification of HLA-DRB1 Gene

[0115] This example is basically the same as Example 1, the only difference is that the 5' end and / or 3' end of the first group of amplification primers has a sequence of less than or equal to 8 nucleotides, in this example, respectively in DRB1 -F2-1, DRB1-F2-2, DRB1-F2-3, DRB1-F2-4, DRB1-F2-5, DRB1-F2-6, DRB1-F2-7 and DRB1-2R 5' end and 3 1 nucleotide is added at the ' end, and the nucleotide can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine), and in this embodiment, the 5' end is increased C, G is added at the 3' end, exon 2 of the HLA-DRB1 gene can be amplified by using the first set of amplification primers, and the 5' end and / or 3' end of the second set of amplification primers is increased by less than or equal to 8 The sequence of 2 nucleotides, in this embodiment, is the sequence of adding 8 nucleotides at the 5' end and 3' end of DRB1-F3 and DRB1-R3 respectively, in this embodiment, the 5' at ...

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Abstract

The invention discloses a primer group, kit and method for HLA-DRB1 gene typing, and belongs to the field of gene detection. According to the primer group for HLA-DRB1 gene high-resolution typing, primers are designed correspondingly according to exons 2 and exons 3 of HLA-DRB1 genes, and the primers are utilized to conduct PCR amplification on the HLA-DRB1 genes. The primers conduct PCR amplification on the HLA-DRB1 genes, thus the gene fragment length of an amplified product is smaller than 1 kb, the requirement for the integrity of a template is reduced, the amplification efficiency is high, the amplification reaction time is short, the cost is reduced, the gene typing requirement of the large-scale sample HLA-DRB1 genes can be met, the more accurate basis is provided for clinical HLA matching, a proper transplant donor is provided for a patient, the rejection reaction in the transplant process is reduced, and the success rate of organ transplantation and the survival rate of the patient are increased.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a primer set, a kit and a method for HLA-DRB1 genotyping. 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 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 to occur rejection reaction. Therefore, efficient and accurate typing of HLA is very...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6881C12Q1/6869C12N15/11
Inventor 康颖
Owner BEIJING NUOSHI KANGYING MEDICAL TECH
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