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Primers, kit and method for quickly typing KIR (Killer Immunoglobulin-like Receptor) genes

A kit and gene technology, applied in the field of genetic engineering, can solve the problems of expensive consumables, inability to detect KIR gene alleles and loci, missed detection and false detection, etc., to achieve low experimental cost, saving operation time and workload. Effect

Active Publication Date: 2014-09-10
SHANGHAI TISSUEBANK BIOTECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have certain limitations, mainly in that PCR-SSOP and RT-PCR methods require specific instruments, and reagents and consumables are expensive
Although, generally speaking, PCR-SSP is a relatively simple, convenient, and highly specific method, and there are currently kits for detecting KIR with single specific primers based on the PCR-SSP method, but because they are all used in the past Therefore, the alleles and loci of KIR genes newly discovered in recent years cannot be detected, and the amplification products are mostly long fragments, and the requirements for the sample genomic DNA are also high. Unable to meet the requirements for obtaining mid-to-high resolution typing results for KIR genes

Method used

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  • Primers, kit and method for quickly typing KIR (Killer Immunoglobulin-like Receptor) genes
  • Primers, kit and method for quickly typing KIR (Killer Immunoglobulin-like Receptor) genes
  • Primers, kit and method for quickly typing KIR (Killer Immunoglobulin-like Receptor) genes

Examples

Experimental program
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Embodiment 1

[0023] Example 1: Preparation of a kit for rapid detection of KIR gene

[0024] 1. Synthesis of primers

[0025] The primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the primer sequences are respectively shown in SEQ ID NO.1-46.

[0026] At the same time, in order to ensure the normal operation of the reaction system, a pair of upstream and downstream primers NCXF (SEQ ID NO.47) and NCXR (SEQ ID NO.48) were also designed as internal control primers, and the size of the amplified fragment was 790bp.

[0027] The specific sequence is shown in Table 1

[0028] Table 1. Primers for rapid detection of KIR genes

[0029]

[0030]

[0031] 2. Preparation of PCR reaction mixture

[0032] Mix primers shown in SEQ ID NO.1-48, dNTPs, dye cresyl red, and PCR buffer. The detection primers KP-01 and KP-02 form a group, which are combined in pairs in order to form 23 primer groups. The concentrations of the detection primers SEQ ID NO.1-46 were all 0.5 μM,...

Embodiment 2

[0033] Example 2: Typing detection of KIR gene in samples

[0034] Positive DNA samples covering 16 KIR genotypes were selected. Add to PCR tubes separately, and add Taq enzyme to each tube at the same time. After adding the sample, mix the reaction mixture evenly, centrifuge briefly, and carry out the PCR reaction.

[0035] The conditions of the PCR reaction 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.

[0036] Use 0.5×TBE buffer to prepare 2% agarose gel. Take 3ul of PCR products and directly spot on the gel well, electrophoresis for 30 minutes, and then take pictures and record under ultraviolet light. For specific gel electrophoresis results, please refer to the attached figure 1 .

[0037] Interpretation of the results: all the results showed bands with the same size as the target fragment, indicating that the kit of ...

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Abstract

The invention belongs to the technical field of gene engineering and discloses primers for quickly typing KIR (Killer Immunoglobulin-like Receptor) genes, a kit containing the primers and a method for quickly typing the KIR genes by adopting the primers. The specific primers of the KIR genes are used to enhance specific combination and can be fully used for detecting 16 known KIR allelic genes at present. In addition, the specific primers, dNTPs (Deoxyribonucleotide), a PCR (Polymerase Chain Reaction) buffer liquid and dye are mixed in advance to greatly save the operating time and the workload, so that the method has the characteristics of quickness, simplicity and convenience, accuracy and intuitiveness. The typing experiment of the whole gene can be completed within 3 hours, so that the problem on quick typing of KIR genes is solved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, kits and methods for rapid typing of KIR genes. Background technique [0002] The killer cell immunoglobulin-like receptor (KIR) gene encodes a family of activating and inhibitory KIR receptors, which are expressed on the surface of NK cells and some T cells, and transduce and activate by specifically recognizing MHC-I molecules on the surface of target cells Or inhibit the signal, thereby regulating the activity of NK cells and T cells, and play an important role in anti-infection, tumor monitoring, transplantation immunity and autoimmune diseases. The KIR gene belongs to autosomal co-dominant inheritance, located on human chromosome 19q13.42, about 100-200kb in length, and highly polymorphic. A total of 16 KIR genes have been identified, including 14 functional genes (KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1) and 2 pseudogenes (KIR2DP1, KIR3DP1), forming c...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2545/101
Inventor 潘捷郑仲征田石磊
Owner SHANGHAI TISSUEBANK BIOTECH
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