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HLA (Human Leukocyte Antigen) gene high-resolution genotyping method based on Illumina GA sequencing technology

A typing method and sequencing technology, applied in the fields of PCR sequencing and nucleic acid sequencing, can solve the problems of complex experimental process, difficult to apply to large-scale HLA high-resolution typing projects, and high cost, and achieve the effect of simplifying experimental operation.

Active Publication Date: 2010-12-22
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The HLA-SBT experimental steps based on the Sanger sequencing method include: PCR amplification, PCR product electrophoresis, PCR product purification, sequencing reaction, sequencing reaction product purification, sequencer sequencing, sequencing result typing, and subsequent addition of GSSP, etc., the entire experiment The process is complicated, and different PCR products cannot be mixed together during the experiment, which greatly increases the workload of the experiment
[0008] The disadvantages of HLA-SBT, such as the complex experimental process, low throughput and high cost, make it difficult to apply to large-scale HLA high-resolution typing projects

Method used

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  • HLA (Human Leukocyte Antigen) gene high-resolution genotyping method based on Illumina GA sequencing technology
  • HLA (Human Leukocyte Antigen) gene high-resolution genotyping method based on Illumina GA sequencing technology
  • HLA (Human Leukocyte Antigen) gene high-resolution genotyping method based on Illumina GA sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] sample extraction

[0052] Using KingFisher automatic extractor (please provide supplier information) (Thermo Company, USA) from 950 blood samples with known HLA-SBT typing results (China Hematopoietic Stem Cell Donor Database (hereinafter referred to as "China Bone Marrow Bank")) Extract DNA. The main steps are as follows: Take out 6 deep-well plates and 1 shallow-well plate matched with the Kingfisher automatic extractor, add a certain amount of matching reagents according to the instructions and mark them, and place all the well-plates with reagents in the For the corresponding position, select the program "Bioeasy_200ul BloodDNA_KF.msz" and press "star" to execute the program for nucleic acid extraction. After the program is over, collect about 100ul of the eluted product in the plate Elution, which is the extracted DNA, which is ready to be used as a template in the next step of PCR.

Embodiment 2

[0054] PCR amplification

[0055]The 950 copies of DNA obtained in the sample extraction step were numbered 1-950 in turn, and divided into 10 groups, with 95 copies of DNA in each group, labeled as HLA-1, HLA-2, HLA-3, HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10. For each group of samples, 95 sets of PCR primers (Table 2) used to amplify HLA-A / B exon 2, 3, and HLA-DRB12 exon with bidirectional primer labels (Table 1) were used to separate Amplify 95 DNA samples. The PCR reaction was carried out in a 96-well plate, with a total of 7 plates, numbered HLA-X-P-A2, HLA-X-P-A3, HLA-X-P-A4, HLA-X-P-B2, HLA-X-P-B3, HLA-X-P- B4 and HLA-X-P-DRB1-2 ("X" indicates the sample group number information 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10, "A2 / 3 / 4, B2 / 3 / 4 , DRB1-2" indicates the site of amplification), a negative control without template was set in each plate, and the primers used in the negative control were PI-1 (Table 1) labeled primers. At the same time as the experiment, record the sample gro...

Embodiment 3

[0080] PCR product pooling and purification

[0081] For the "X" group ("X" is 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10) samples, from the remaining PCR products of HLA-X-P-A2 in the 96-well plate (negative control Except) each take 20ul and mix them in a 3ml EP tube, marked as HLA-X-A2-Mix, do the same for the other 6 96-well plates of the samples of the "X" group, marked as HLA-X- A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4-Mix and HLA-X-D2-Mix, shake and mix, from HLA -X-A2-Mix, HLA-X-A3-Mix, HLA-X-A4-Mix, HLA-X-B2-Mix, HLA-X-B3-Mix, HLA-X-B4-Mix, and HLA-X - Take 200ul from each of D2-Mix and mix in a 3ml EP tube, marked as HLA-X-Mix. Each of 500ul of DNA mixture was taken from the column and purified by Qiagen DNA Purificationkit (see the instruction manual for details of the specific purification steps), and the DNA concentrations of the purified 200ul of DNA obtained by Nanodrop 8000 (Thermo Fisher Scientific) were as follows:

[0082]

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Abstract

The invention provides an HLA (Human Leukocyte Antigen) gene high-resolution genotyping method based on an Illumina GA sequencing technology and a primer label for the method.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to the technical field of PCR sequencing. In addition, the present invention also relates to DNA molecular labeling technology and incomplete DNA breaking strategy. The method of the present invention is particularly applicable to the second generation sequencing technology. The method of the invention relates to a typing method of DNA sequence, especially a high-resolution typing method of HLA gene. Background technique [0002] Human leukocyte antigen, namely HLA (human leukocyte antigen, HLA), is one of the most polymorphic gene systems found so far. It is closely related to the rejection of allogeneic organ transplantation. The study found that, at the time of transplantation, the higher the degree of HLA-related gene matching between the donor and the recipient, the higher the resolution and the longer the survival time of the graft. [0003] HLA-SBT (Sequen...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6853C12Q1/6869C12Q1/6881C07H21/00C12Q2600/156C12Q2600/16
Inventor 李剑田埂蒋慧章文尉
Owner BGI GENOMICS CO LTD
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