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PCR-SBT method for HLA genotyping and reagent thereof

A technology of PCR-SBT and HLA-B, which is applied in the field of genotyping detection methods and its reagents, can solve the problems affecting clinical work and the inability to specify alleles, so as to improve the level of organ transplantation, high throughput of operation results, Reduce the effect of rejection

Active Publication Date: 2010-12-29
浙江省血液中心
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Problems solved by technology

However, the current PCR-SBT typing method for HLA-A, B, and C sites is mainly aimed at determining the genotypes of exons 2, 3, and 4, which are relatively concentrated in polymorphic sites. Exons 5, 6, and 7 are not sequenced. Due to the high degree of genetic polymorphism in HLA, there are also certain polymorphisms in exons 1, 5, 6, and 7. Exon polymorphism may cause partial alleles to be unassigned, ambiguous or wrong results, which seriously affect clinical work

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  • PCR-SBT method for HLA genotyping and reagent thereof
  • PCR-SBT method for HLA genotyping and reagent thereof
  • PCR-SBT method for HLA genotyping and reagent thereof

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Embodiment 1

[0076] This implementation specifically takes leukemia patients to carry out HLA-A, HLA-B and HLA-C genotyping as an example to describe the content of the present invention in detail. A kind of HLA-A, HLA-B and HLA-C gene classification used in the present invention The PCR-SBT method of type specifically comprises the following steps:

[0077] 1. Prepare human genomic DNA as a template for PCR amplification in subsequent steps.

[0078] Take 200 μL of whole blood to be tested, and extract genomic DNA according to the instructions of the invitrogen DNA isolation kit (other methods can be used to extract genomic DNA), and use a spectrophotometer to measure the concentration and purity of genomic DNA.

[0079] 2. Synthesize 6 pairs of amplification primers and 36 sequencing primers, and dilute the amplification primers to 50 μM with pure water. The amplification primers and sequencing primers are described in the content of the invention, and will not be repeated here.

[0080...

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Abstract

The invention provides a PCR-SBT method for HLA genotyping. Exons 1, 2, 3, 4, 5, 6, 7 of an HLA-A locus, an HLA-B locus and an HLA-C locus are subjected to genotyping. The method comprises the following steps of: (1) preparing human genomic DNA; (2) designing an amplification primer; (3) performing double digestion purification on an amplification product; (4) designing a sequencing primer and performing sequencing PCR on a purification product; (5) purifying a sequencing product by a sodium acetate-ethanol precipitation method and performing capillary electrophoresis sequencing; and (6) performing software analysis on the acquired sequence to determine genotypes thereof. The exons 1, 2, 3, 4, 5, 6, 7 of the HLA-A, B, C loci are subjected to full-length sequence sequencing, so that the full length of the related exons of the HLA-A, B, C loci and part of intron oligonucleotide sequences are obtained and the HLA genotyping is accurately performed.

Description

technical field [0001] The invention relates to a genotyping detection method and a reagent thereof, in particular to a molecular biology detection method and a reagent thereof for genotyping of exons 1-7 of HLA-A, B, and C sites. Background technique [0002] The human leukocyte antigen (HLA) gene is located in the 21.3 region of the short arm of chromosome 6. It is the main gene system that regulates the specific immune response of the human body and is the most polymorphic genetic system known so far. HLA antigens are closely related to the rejection of allogeneic organ transplantation, and the survival of the graft after organ transplantation largely depends on whether the HLA types of the donor and the recipient are compatible. HLA locus typing is of great significance for selecting suitable donors, reducing the incidence of graft-versus-host disease (GVHD), and improving the survival rate of grafts. [0003] HLA-A, -B, -C locus genes are classic HLA class I genes with...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 朱发明章伟严力行
Owner 浙江省血液中心
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