Primers, kit and method for high-resolution typing of HLA gene

A technology for high-resolution, typing methods, applied in the field of genetic engineering

Inactive Publication Date: 2018-05-18
SHANGHAI TISSUEBANK MEDICAL LAB CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the random sequencing error of the sequencer and the errors generated in the steps of comparison and assembly in the analysis of biological information, the above products and technologies all have a certain error rate.

Method used

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  • Primers, kit and method for high-resolution typing of HLA gene
  • Primers, kit and method for high-resolution typing of HLA gene
  • Primers, kit and method for high-resolution typing of HLA gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design and synthesis of primers

[0052] In this example, the HLA exon sequences required for PCR primer design are sourced from IMGT / HLA Database, website: http: / / www.ebi.nc.uk / ipd / imgt / hla / . The primers were designed using Primer Premier 6.0 software, and the designed primers were compared in the IMGT database to confirm that the set of primers could specifically amplify or sequence the desired fragments.

[0053] The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the specific sequences were as described above.

Embodiment 2

[0054] Embodiment 2: Screening of amplification primers

[0055] 1. Randomly select 96 DNA samples (including a negative control) to amplify the exons of HLA genes, and preliminarily judge the amplification efficiency and specificity of the primer set by agarose gel electrophoresis.

[0056] (1) DNA extraction was performed according to the QIAamp DNA Blood Mini kit, followed by DNase-FreeddH 2 O was diluted to 10ng / μL for later use.

[0057] (2) Use 25μL amplification system: 2.5μL 10×Vazyme Buffer (Mg 2+ plus); 4μL 2.5mM dNTPs; 1μL 10μM primer set; 5μL 5×PCR Enhancer; 3μL 10-50ng / μL sample DNA; 1μL Vazyme DNA Polymerase (5U / μL); use DNase-Free ddH 2 O complements the reaction system.

[0058] (3) For the amplification reaction of the primer sets HLA-A-f / HLA-A-r, HLA-B-f / HLA-B-r and HLA-C-f / HLA-C-r, the reaction condition is 95°C for 2min; then run 35 cycles according to the following procedure , 93°C for 30s, 60°C for 30s, 72°C for 180s; store at 15°C. For the ampli...

Embodiment 3

[0064] Embodiment 3: Illumina MiSeq and Illumina NextSeq500 on-board key program settings

[0065] 1. Illumina MiSeq Samplesheet settings

[0066] Refer to attached Figure 4 , open ILLUMINA EXPERIMENT MANAGER software, fill in ReagentCartridge Barcode in turn, select Sample Prep Kit as "Nextera IT v2", select Index Reads as "2", fill in Experiment Name, select Read Type as "Paired End", fill in Cycle Read 1 and Cycle Read2 is 151, select "Use Adapter Trimming" on the right interface, and click Next.

[0067] In the Sample Sheet Wizard-Sample Selection interface, add Index information for each sample according to the actual situation, and click Finish after adding.

[0068] 2. Key program settings on Illumina NextSeq500

[0069] Refer to attached Figure 5 , follow the prompts on the machine to the Run Setup step, enter Run Name, LibraryID in turn, select "Paired End", and fill in "151", "151", "8", and "8" in Read Length from left to right , click Next and follow the sub...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer combination for high-resolution typing of HLA gene, a kit containing the primer combination, and a method for high-resolution typing of HLA gene. Compared to the traditional methods such as SBT, SSO and the like, the method of the present invention has the following advantages that the sequencing reaction cansimultaneously determine 480 samples so as to provide high-throughput characteristic, the HLA typing results obtained by using the method of the present invention contain the low-resolution typing results and the high-resolution typing results so as to provide high-resolution characteristic, and the HLA typing detection results of 3000 random samples show that the typing accuracy can achieve 100%so as to provide high accuracy characteristic.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, kits and methods for high-resolution typing of HLA genes. Background technique [0002] The Human Leukemia Antigen (HLA) gene is the human major histocompatibility complex MHC. The HLA gene is located on the short arm of chromosome 6. It is the main system that regulates the specific immune response of the human body and determines individual differences in disease susceptibility. , is closely related to the rejection of allogeneic hematopoietic stem cell transplantation and organ transplantation. Allogeneic hematopoietic stem cell transplantation is one of the main treatments for leukemia, aplastic anemia, myelodysplastic syndrome and other malignant blood diseases. The matching degree of HLA genotype is the main factor determining the long-term survival of grafts. HLA typing was performed on the patients. [0003] HLA is characterized by a high degree of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6881C12N15/11
CPCC12Q1/6881C12Q2600/156
Inventor 郑仲征廖宽镇邓佳刘娴王莉萍
Owner SHANGHAI TISSUEBANK MEDICAL LAB CO LTD
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