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Primer group, kit and method for amplification of human leukocyte antigen-B (HLA)-B gene, and primer group, kit and method for genotyping of HLA-B gene

An HLA-B, gene amplification technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of high DNA template integrity, full-length fragment length, and high cost

Active Publication Date: 2018-09-14
BEIJING NUOSHI KANGYING MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Primer group, kit and method for amplification of human leukocyte antigen-B (HLA)-B gene, and primer group, kit and method for genotyping of HLA-B gene
  • Primer group, kit and method for amplification of human leukocyte antigen-B (HLA)-B gene, and primer group, kit and method for genotyping of HLA-B gene
  • Primer group, kit and method for amplification of human leukocyte antigen-B (HLA)-B gene, and primer group, kit and method for genotyping of HLA-B gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1 Amplification of HLA-B gene

[0095] 1. Sample DNA extraction

[0096] DNA was extracted from 96 blood samples with known HLA-B 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.

[0097] 2. Design HLA-B gene amplification primers

[0098] According to the latest HLA-B gene sequence in the IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), such as figure 1 As shown, the HLA-B gene has 8 exons, multiple groups (two for each group) of suitable conserved regions are searched, and each group of conserved regions is guaranteed to cover the 8 exons of the HLA-B 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 allele group-specif...

Embodiment 2

[0110] Embodiment 2 Amplification of HLA-B gene

[0111] 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, an...

Embodiment 3

[0112] Embodiment 3 Amplification of HLA-B gene

[0113] 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, G is added at the 3' end, and exons 1-3 of the HLA-B gene can be amplified by 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 example, the 5'→3' at the 5' end increases CACAGTGT, and the 5'→3' at t...

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Abstract

The invention discloses a primer group, a kit and a method for amplification of a human leukocyte antigen-B (HLA)-B gene, and the primer group, the kit and the method for genotyping of the HLA-B gene,belonging to the field of gene detection. According to the primer group, provided by the invention, for the amplification of the HLA-B gene, at least two sets of primers are designed according to theeight exon regions of the HLA-B gene; the primers are used for performing polymerase chain reaction (PCR) amplification on the HLA-B gene, so that a product having a gene fragment length of less than1.5 kb is obtained; after the HLA-B gene is subjected to the PCR amplification by the primers, the gene fragment length of the amplified product is less than 1.5 kb, so that the requirement for template integrity is reduced, the amplification efficiency is high, amplification reaction time is short, and the cost is low. The primer groups, the kits and the methods can meet the needs of amplification or genotyping of the HLA-B gene in mass samples, provide a more accurate basis for clinical HLA matching, provide suitable transplant donors for patients, reduce the rejection in a transplantationprocess and improve the success rate of organ transplantation and the patient survival rate.

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-B gene amplification and 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 t...

Claims

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Application Information

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