Primer, kit and method related to determination of full-length sequence of human leukocyte antigen HLA-E, -F and-G genes

A leukocyte antigen, HLA-E technology, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the problems of expensive and limited large-scale application of HLA gene sequencing and typing

Inactive Publication Date: 2020-02-14
SHENZHEN BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high cost of commercial kits, the large-scale application of HLA gene sequencing typing in population genetics and bone marrow banking is limited

Method used

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  • Primer, kit and method related to determination of full-length sequence of human leukocyte antigen HLA-E, -F and-G genes
  • Primer, kit and method related to determination of full-length sequence of human leukocyte antigen HLA-E, -F and-G genes
  • Primer, kit and method related to determination of full-length sequence of human leukocyte antigen HLA-E, -F and-G genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] This example provides a complete set of full-length sequences of HLA-E 3.8kb, HLA-F 3.5kb and HLA-G 3.1kb alleles.

[0085] Take HLA-E gene full-length amplification primers E-Genome-F and E-Genome-R in the present invention, HLA-F gene full-length amplification primers F-Genome-F and F-Genome-R, HLA-G Gene full-length amplification primers G-Genome-F and G-Genome-R were used to amplify the full-length HLA-E, -F, and -G genes of 8 random Asian DNA samples respectively, and carried out in ABI 9700 PCR instrument. The amplification reaction system consists of:

[0086]

[0087] In the HLA-E full-length sequence amplification reaction system, the cycle parameters are:

[0088]

[0089] In the HLA-F full-length sequence amplification reaction system, the cycle parameters are:

[0090]

[0091]

[0092] In the HLA-G full-length sequence amplification reaction system, the cycle parameters are:

[0093]

[0094] Take 2 μL of PCR products, stain with cyber-gre...

Embodiment 2

[0128] This example presents the experimental results related to the whole gene sequencing of HLA-E in Example 1.

[0129] 2.1 Identification of HLA-E whole gene PCR amplification products

[0130] Using the PCR amplification primers designed in this paper, the entire HLA-E gene fragments of 16 samples were successfully amplified, and the length of the PCR product was about 3.6Kb. Take 5 μL and electrophoresis in 1.2% agarose gel, and a clear amplification band can be seen. The Marker is DL10000 DNA Marker (Takara, Dalian Baosheng Company). Amplification results see figure 1 . figure 1 The upper and middle figures show the electropherograms of the amplification products of Marker and samples C1-C8 respectively, and the lower figures show the electrophoresis figures of the amplification products of Marker and samples C9-C16 respectively.

[0131] 2.2 Sequencing Peak Map and Splicing Effect

[0132] Using the step-by-step sequencing primers ESF1-ESF7 designed in this paper, ...

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Abstract

The invention discloses a primer, a kit and a method related to determination of full-length sequence of human leukocyte antigen HLA-E, -F and-G genes. By adopting the primer and the method, whole HLA-E, -F and-G genes are amplified with clear bands, specific amplification, and good quality of peaks of sequencing results. The whole HLA-E, -F, -G gene sequences and typing results can be obtained bysplicing the sequences detected by walking primers.

Description

technical field [0001] The invention relates to biomedicine and immunogenetics, in particular to a primer, a kit and a method related to the determination of the full-length sequences of human leukocyte antigens HLA-E, -F, and -G genes. Background technique [0002] Human leukocyte antigen (Human Leukocyte Antigen, HLA) 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. The HLA system is divided into 3 categories: HLA-I, HLA-II and HLA-III. HLA class I genes can be divided into classical MHC class I (HLA-Ia) and nonclassic MHC class I (HLA-Ib) genes according to the tissue distribution, function and polymorphism of the encoded products. HLA-E, -F, -G genes all belong to HLA-Ib class, they are very similar in structure, compared wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6881C12N15/11
CPCC12Q1/6881C12Q1/6883C12Q2600/156
Inventor 徐筠娉洪文旭王宋兴
Owner SHENZHEN BLOOD CENT
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