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Primers, kit and method for KIR genotyping

A genotyping and kit technology, applied in the field of genetic engineering, can solve the problems of long time, cumbersome operation, and high cost, and achieve the effects of short time-consuming, low experimental cost, and easy operation

Inactive Publication Date: 2021-05-04
SHENZHEN TISSUEBANK PRECISION MEDICINE CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the existing methods for KIR typing by sequencing (sequence based typing, SBT) are costly, cumbersome and time-consuming

Method used

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  • Primers, kit and method for KIR genotyping
  • Primers, kit and method for KIR genotyping
  • Primers, kit and method for KIR genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Design and synthesis of primers

[0036] In this example, the KIR exon sequence required for PCR primer design is derived from the KIR Database, website: https: / / www.ebi.ac.uk / ipd / kir / .

[0037] The primers were designed using Primer Premier 6.0 software, and the designed primers were compared in the KIR database to confirm that the set of primers could specifically amplify the desired fragment.

[0038] The primers were synthesized by Shanghai Bioengineering Technology Co., Ltd., and the specific sequences are listed in Table 1.

Embodiment 2

[0039] Embodiment 2: Screening of amplification primers

[0040] Randomly select 96 DNA samples (including a negative control), all of which come from the clinical research service samples of Shanghai Dishuo Beiken Gene Technology Co., Ltd., amplify the KIR gene exons, and judge by agarose gel electrophoresis Amplification efficiency and specificity of primer sets.

[0041] 1. DNA extraction

[0042] DNA extraction was performed according to the QIAampDNABloodMinikit kit followed by DNase-Free ddH 2 O was diluted to 10-30ng / μL for later use.

[0043] 2. PCR amplification system

[0044] Use 10 μL of the amplification system: 5 μL 2*GC Buffer Ⅰ (stored at 4°C) (TAKARA, Cat. No. RR02AG); 1 μL 2.5 mM dNTPs (TakaraBio); 0.4 μL Control (2.5p); 1 μL primer set (primers were synthesized by Shanghai Sangon) ); 1μL cresyl red (1mg / ml, stored at 4°C); 1μL 10-30ng / μL sample DNA; 0.07μL La Taq enzyme (stored at -20°C) (TAKARA, Cat. No. RR02MA); use DNase-Free ddH 2 O complements the ...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer combination for KIR gene typing, a kit containing the primer combination and a method for typing a KIR gene by adopting the primer combination or the kit. By adopting the primer combination disclosed by the invention, all sites of the KIR gene can be amplified by using 16 amplification tubes, and all types which can be distinguished by virtue of exon sequences can be detected under the condition that exon reference sequences are complete.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, kits and methods for KIR genotyping. Background technique [0002] Organ transplantation is the transplantation of healthy organs into recipients to replace and rapidly restore function of organs that have been disabled by fatal diseases. Bone marrow transplantation is to inject normal bone marrow into the patient's body through veins to replace the diseased bone marrow, rebuild the patient's hematopoietic and immune functions, and achieve the purpose of treating various blood diseases including leukemia. In bone marrow or organ transplantation, rejection has been a major factor affecting graft survival. For a long time, the medical community generally believed that human leukocyte antigen (human leukocyte antigen, HLA) plays a decisive role in the recognition of alloantigens in rejection. However, with the deepening of research, it was found that some pati...

Claims

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Application Information

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IPC IPC(8): C12Q1/6881C12Q1/686C12N15/11
CPCC12Q1/6881C12Q1/686C12Q2600/156
Inventor 郑仲征杜可明徐祥袁志阳李岱阳
Owner SHENZHEN TISSUEBANK PRECISION MEDICINE CO LTD
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