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Human leukocyte antigen HLA-A gene sequencing and typing method

A technology of gene sequencing and typing methods, which is applied in the fields of biomedicine and immunogenetics, and can solve problems such as ambiguity

Inactive Publication Date: 2011-08-31
SHENZHEN BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to systematically sequence and type the coding regions of HLA-A and -B genes, use group-specific primers to sequence and type the second and third exons of HLA-DQB1 and DRB1 to solve the problem of HLA sequencing and typing. Ambiguous result issues that arise

Method used

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  • Human leukocyte antigen HLA-A gene sequencing and typing method
  • Human leukocyte antigen HLA-A gene sequencing and typing method
  • Human leukocyte antigen HLA-A gene sequencing and typing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0176] This example provides a complete set of schemes for obtaining 38 HLA-A 4.3kb full-length alleles and 30 HLA-B 3.7kb full-length sequences.

[0177] First, 45 samples containing common alleles in all sublines of HLA-A were selected from 1000 blood samples of Shenzhen hematopoietic stem cell donors who had undergone high-resolution sequencing of HLA-A and -B genes, and the results were known. 38 samples containing common alleles in all sublines of HLA-B were subjected to the gene sequence determination of the present invention. Take HLA-A gene full-length amplification primers A-Genome-F and A-Genome-R in the present invention, carry out HLA-A gene full-length amplification to 45 samples, take HLA-B gene full-length amplification primer For B-Genome-F and B-Genome-R, the full-length HLA-B gene was amplified on 38 samples, and the amplification was carried out in ABI 9700 PCR instrument. The amplification reaction system consisted of:

[0178] 10×Pfu Buffer 2.5μL

[0179...

Embodiment 2

[0195] This example gives an example of performing HLA-A and -B high-resolution sequencing typing on 384 cases of voluntary hematopoietic stem cell donor samples in Shenzhen.

[0196] The applicant randomly selected 384 samples with known HLA-A, -B allele types from hematopoietic stem cell voluntary donors in Shenzhen, and used the PCR primers and sequencing primers of the present invention to perform sequencing typing to verify the present invention implementation effect. Using the amplification and sequencing primers of the present invention to perform sequencing and typing experiments on the above samples, clear HLA-A and -B genotypes were finally obtained and the ambiguous results in the selected samples could be resolved.

[0197] Firstly, two pairs of PCR amplification primers are used to perform PCR amplification reaction on HLA-A and -B of the sample respectively, wherein, the first pair of amplification primers amplifies the first exon to the fourth exon of the two ge...

Embodiment 3

[0235] In order to verify the effectiveness of the HLA-DRB1 group-specific primers, in this example, 8 samples containing 8 group-specific alleles ( DR01, DR02 / 15 / 16, DR03 / 11 / 13 / 14, DR04, DR07, DR08 / 12, DR09, DR10) specimens, respectively with the 5' end group specific Primers and 3'DR-generic amplify the sequence from exon 2 to exon 3 of DRB1. The amplification reaction system used is:

[0236] 10×Buffer 12.5μL

[0237] MgCl 2 (25mM) 1.5μL

[0238] dNTP (25mM) 2.0μL

[0239] 1 μL each of PCR primers (10 μM )

[0240] Genomic DNA 100ng

[0241] Clontech LA enzyme (5U / μL) 0.5μL

[0242] ddH 2 0 to 25 μL.

[0243] The amplification reaction procedure used was:

[0244] 95℃ 2min

[0245] 95℃ 15Sec

[0246] 65℃ 15Sec

[0247] 72℃ 2min, 5 cycles

[0248] 95℃ 15Sec

[0249] 60℃ 15Sec

[0250] 72℃ 2min, 26 cycles

[0251] 95℃ 5Sec

[0252] 55℃ 1min

[0253] 72°C 3min, 4 cycles

[0254] 72°C 15min

[0255] 4°C Infinite.

[0256] Take 5 μL of the PCR product, stain...

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Abstract

The present invention discloses a human leukocyte antigen HLA-A and HLA-B gene full-length sequencing method and an HLA gene sequencing and typing method. The HLA-A and HLA-B gene full-length sequencing method comprises the following steps of: a, performing PCR amplification on about 4kb full-length sequences of HLA-A and HLA-B genes by using a pair of primers respectively; and b, cloning the amplification products to a pGEM-Teasy vector, sequencing the full-length sequences by using ten walking sequencing primers in positive and negative directions respectively, and totally obtaining 38 allele 4.3kb full-length sequences of the HLA-A and 30 allele 3.7kb full-length sequences of the HLA-B. The HLA-A and HLA-B sequencing and typing method comprises the following steps of: performing PCR amplification on typing target areas of the HLA-A and HLA-B by using two pairs of primers respectively; and performing two directional sequencing on products by using fourteen sequencing primers respectively, wherein the HLA-DRB1 and HLA-DQB1 sequencing and typing method comprises the following steps of: amplifying sequences of second and third exons of DRB1 and DQB1 by adopting group specificity primers respectively; performing two directional sequencing on the second and third exons of the DRB1 by adopting eight group specificity primers and three sequencing primers; and performing two directional sequencing on the second and third exons of the DQB1 by adopting four sequencing primers respectively.

Description

【Technical field】 [0001] The invention relates to biomedicine and immunogenetics, in particular to the determination of the full-length sequence of the tissue-matching related genes HLA-A and -B and the HLA gene sequencing and typing method. 【Background technique】 [0002] Human Leukocyte Antigen HLA (Human Leukocyte Antigen) molecules present antigenic peptides to T lymphocytes for recognition, and play an extremely important role in adaptive immune responses. HLA gene is also an important genetic structure of human beings, and it is widely used in forensic identification, organ transplant matching and other fields. [0003] As we all know, a high degree of polymorphism is a very important feature of HLA gene. Previous studies on polymorphisms of HLA genes mainly focused on exons encoding antigen-binding peptides (exons 2-4 of HLA-A and -B genes, exons 2 and 4 of HLA-DQB1 and -DRB1 genes). Exon). Many researchers have done a lot of work on these exon typing techniques, d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐筱娉邓志辉王大明高素青
Owner SHENZHEN BLOOD CENT
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