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Primers, kit and method for HLA (human leukocyte antigen) genotyping

A genotyping method and genotyping technology, applied in the field of genetic engineering, can solve the problems of high cost, long turnaround time, cumbersome detection steps of PCR-SBT method, etc., achieve wide coverage, reduce experimental cost, and improve typing efficiency. and the effect of accuracy

Inactive Publication Date: 2016-08-17
SHANGHAI TISSUEBANK BIOTECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCR-SBT method is cumbersome, expensive and has a long turnaround time

Method used

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  • Primers, kit and method for HLA (human leukocyte antigen) genotyping
  • Primers, kit and method for HLA (human leukocyte antigen) genotyping
  • Primers, kit and method for HLA (human leukocyte antigen) genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Design of PCR primers for HLA genotyping

[0056] In this example, all HLA alleles required for PCR primer design are derived from the IMGT / HLA database (Release 3.15.0, 2014-01-17), and its specific website is: http: / / www.ebi.nc.uk / ipd / imgt / hla / .

[0057] The primers were designed manually, and the designed primers were compared and analyzed in the IMGT / HLA database to confirm that the set of primers can specifically bind to all HLA alleles in the group without amplifying other alleles. In the typing method of the present invention, the key is to specifically amplify the target group of HLA genes with the set of primers in the environment of the PCR buffer system, that is, the set of primers has "sequence specificity", and the typing method is sequence-specific Sexual SBT (GSA-SBT).

[0058] In addition, in this example, when designing the PCR primers, a specific design of mismatched bases was also introduced at the 3' end of the downstream primers, ther...

Embodiment 2

[0060] Example 2: Preparation of a kit for HLA genotyping

[0061] Entrust Shanghai Yingjun Biotechnology Co., Ltd. to synthesize all PCR primers GSA1-GSA156 of the present invention.

[0062] Mix the synthesized primers, dNTPs, and Taq enzyme buffer to prepare a PCR reaction mixture. Wherein, the concentration of detection primers is 0.4uM, and the concentration of dNTPs is 0.2mM. In the Taq enzyme buffer, the concentration of Tris-HCL was 10 mM, and the concentration of magnesium chloride was 2.5 mM.

[0063] The PCR reaction mixture, Taq enzyme, etc. are subpackaged and packaged to form the kit of the present invention.

Embodiment 3

[0064] Example 3: Type detection of HLA genes in samples

[0065] The kit of the invention can be used as supplement and verification for ambiguous situations in the PCR-SBT typing results, and can also be used for typing detection of HLA genes of unknown DNA samples.

[0066] Four ambiguous DNA samples with known HLA genotypes of A02 / A11 were selected. Use all the primers of the HLA-A sites designed in the present invention to perform PCR amplification on the sample DNA respectively. After adding the sample, mix the reaction mixture evenly and centrifuge briefly to carry out the PCR reaction.

[0067] The PCR reaction conditions were: 94°C for 5 minutes; followed by 30 cycles of 94°C for 1 minute, 65°C for 2 minutes, and 72°C for 1 minute; finally, 72°C for 10 minutes, then cooled to 15°C for gel electrophoresis detection.

[0068] Use 0.5×TBE buffer to prepare 2% agarose gel. Take 3ul of PCR product and directly spot on the gel well, electrophoresis at 150V for 40mins, an...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer combination for HLA (human leukocyte antigen) genotyping, a kit containing the primer combination and a method for HLA genotyping. Thus, the sequence-specific HLA genotyping (GSA-SBT) technique is quick, simple, accurate and visual. The primer combination can be synchronously used for amplification and sequencing with the commercialized kit, thereby lowering the experimentation cost. Besides, the specific design of mismatched bases are introduced to the 3' terminal of the primer, thereby enhancing the specificity of the primer amplification and ensuring the accuracy of the typing result.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, kits and methods for HLA genotyping. Background technique [0002] The incidence of leukemia in our country is about 6 / 100,000, and bone marrow transplantation has become the only possible treatment for various blood diseases headed by leukemia. At present, acute lymphoblastic leukemia has become a malignant tumor that can be cured. The 5-year disease-free survival rate (EFS) is 70-80%. It is also one of the malignant tumors with the best curative effect and the highest cure rate. Among them, the important role of bone marrow transplantation can be seen. [0003] With the continuous deepening of human genomics and molecular biology research, the success of bone marrow transplantation has a clear correspondence with the matching degree of donor and recipient leukocyte antigen HLA genotypes. The genetic region of the HLA complex is located on the short arm of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 郑仲征杜金伟张凯廖宽镇潘捷杜可明
Owner SHANGHAI TISSUEBANK BIOTECH
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