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The method of hla-dqb1 genotyping and related primers

A HLA-DQB1 and genotyping technology, applied in the field of molecular biology, can solve the problems of short read length of new sequencing technology, PCR product length should not exceed 700bp, DNA length should not be too long, etc.

Active Publication Date: 2015-07-29
BGI GENOMICS CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the first-generation sequencing technology (sequencing technology based on the principle of Sanger sequencing), the DNA length that can be used for the preparation of the sequencing library of the new sequencing technology cannot be too long (currently, the maximum applicable length of Illumina Solexa is 700bp), and The read length of the new sequencing technology is generally short, and the current IllumiaGA bidirectional read length can reach a maximum of 300bp
[0008] In view of the characteristics of the new sequencing technology, the length of the PCR product should not exceed 700bp, and the PCR primers for HLA-SBT based on the original Sanger sequencing method are no longer applicable

Method used

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  • The method of hla-dqb1 genotyping and related primers
  • The method of hla-dqb1 genotyping and related primers
  • The method of hla-dqb1 genotyping and related primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: HLA-DQB1 genetic typing using second-generation sequencing technology (Illumina Solexa)

[0037] 1. sample extraction

[0038] DNA was extracted from 94 blood samples with known HLA-SBT typing results (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "China Bone Marrow Bank")) using KingFisher automatic extractor (Thermo Company, USA). 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_200μl Blood DNA_KF.msz" and press "star" to execute the program for nucleic acid extraction. After the program is finished, about 100 μl of the eluted product collected in the plate Elution is the extracted DNA.

[0039] 2. PCR amplification

[0040] Different PCR index pr...

Embodiment 2

[0091] Example 2. HLA-DQB1 Genotyping Using Sanger Sequencing

[0092] 1. Sample DNA Extraction

[0093] Similar to that described in Example 1, the DNA of 20 known HLA genotypes was extracted from 94 samples with the KingFisher automatic extractor.

[0094] 2. PCR amplification

[0095] Using the DNA extracted by the above-mentioned KingFisher automatic extractor as a template, use 2 pairs of PCR primers, Q-F2 and Q-R2, Q-F3 and Q-R3, for single-tube PCR amplification. The PCR program for each pair of primers is as follows: 96°C 2 minutes; 95°C for 30 seconds → 56°C for 30 seconds → 72°C for 20 seconds (35 cycles); 15°C∞.

[0096] The PCR reaction system for HLA-Q is as follows:

[0097] Promega 5×buffer I (Mg2+plus)

5.0μl

dNTP mix (2.5mM each)

2.0μl

Primer mix (25ng / μl)

3.0μl

PromegaTaq (5U / μl)

0.2μl

[0098] DNA (about 20ng / μl)

2.0μl

wxya 2 o

12.8μl

total

25.0μl

[0099] ...

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Abstract

A method for amplifying exons 2 and / or 3 of HLA-DQB1 gene and the specific primers used in the method are provided. And a method for genotyping HLA-DQB1 gene and the amplification primer pairs used in the method are also provided.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular, the invention relates to a method for genotyping HLA-DQB1 and specific primers used in the method. 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. According to the characteristics of HLA gene structure and distribution, it is divided into three types of molecules: HLA-Ⅰ, HLA-Ⅱ and HLA-Ⅲ. Among them, the functions of HLA-Ⅰ and HLA-Ⅱ molecules are mainly related to immune rejection. The molecular functions are mainly related to the synthesis of part of the immune-related complement system and inflammation-related factors. 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/172C12Q2600/16C12Q2600/156C12Q1/6881
Inventor 李剑张现东刘莹
Owner BGI GENOMICS CO LTD
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