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Method for amplifying and typing HLA gene and relevant primer thereof

An amplification primer and gene amplification technology, applied in the field of molecular biology, can solve the problems of high cost, long time, and inability to distinguish, and achieve the effect of saving time and cost, high accuracy and resolution

Active Publication Date: 2010-02-24
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some technical problems in the existing SBT method. For example, most of its PCR primers amplify exons 2, 3, and 4 of HLA-A, B, and C sites. When the nucleotide difference is outside the amplification area, it cannot be distinguished; there are too many PCR reactions, the cost is high, and the time is too long, etc.

Method used

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  • Method for amplifying and typing HLA gene and relevant primer thereof
  • Method for amplifying and typing HLA gene and relevant primer thereof
  • Method for amplifying and typing HLA gene and relevant primer thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1. Using the HLA typing system of the present invention to perform typing detection on samples with known genotypes.

[0041] In this example, high-resolution genotyping of HLA-A, B, and C loci was performed on 20 human blood samples with known HLA genotypes using the PCR amplification primer pair and sequencing primers of the present invention. The specific steps of the test are as follows:

[0042] 1. Genomic DNA was extracted by salting out method.

[0043] The test steps are as follows:

[0044] (1) Operate in a biological safety cabinet, draw 200 μl of mixed whole blood into a 2ml EP tube, and close the biological safety cabinet according to the procedure after the operation.

[0045] (2) Add 700 μl of erythrocyte lysate into the EP tube, shake for 15 seconds, and centrifuge at 14,000 rpm for 1 min.

[0046](3) Discard the supernatant, put the opening of the EP tube downward on the absorbent paper, and drain the remaining liquid as much as possible.

[...

Embodiment 2

[0085] Example 2. Discovery of novel alleles using the system of the invention.

[0086] In this example, multiple human blood samples of unknown genotypes were genotyped using the primers of the present invention, and the method used was as described in Example 1. After sequencing, it was found that the genotype of one of the samples was different from the existing genotypes recorded in the database (for the sequencing results, see Figure 5 ), as shown by the circle in the figure, the nucleotide at the 695th position of exon 4 of the HLA-C gene of this sample is G, while the nucleotide at this position in the database is T, so this sample is A new allele of HLA-C. This proves that new allele types can be found by using the method of the present invention.

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Abstract

The invention provides a method for amplifying an HLA gene and a specificity primer pair used by the method and also provides a method for typing the HLA gene and an amplifying primer pair and a sequencing primer pair which are used by the method.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular, the present invention relates to methods for amplification and typing of HLA-A, HLA-B and HLA-C genes, and specific primers used in the methods. Background technique [0002] Human leukocyte antigen, or HLA (human leukocyte antigen), is the main gene system that regulates the body's specific immune response and determines individual differences in disease susceptibility, and is closely related to the rejection of allogeneic organ transplantation. [0003] The HLA system plays an important role in antigen recognition, antigen presentation, immune response and regulation, and destruction of foreign antigen target cells, and is the main material basis for immune rejection. Both class I and class II antigens on the cell surface of grafts are strong transplantation antigens. Humoral immunity and cellular immunity are both involved in the rejection of grafts. Whether it is transp...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68C12N15/11
CPCC12Q1/6881C12Q2600/156C12Q2600/16
Inventor 李剑瞿京辉杜金伟王莉萍张红云田中明黄常健许婷婷沈苗忠杨华志甄贺富陈浩潘捷余艳华陈演华廖宽震姜范波华桑刘丹叶葭高扬杨玲
Owner BGI GENOMICS CO LTD
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