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Polymerase chain reaction-sequence based typing (PCR-SBT) method and kit of MHC class I chain-related gene B (MICB)

A PCR-SBT, MICB-F technology, applied in the field of molecular biology, can solve the problem that there is no MICB genotyping reagent, and achieve the effect of improving resolution and accuracy, good stability and reducing cost

Inactive Publication Date: 2013-02-06
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no MICB genotyping reagents on the market

Method used

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  • Polymerase chain reaction-sequence based typing (PCR-SBT) method and kit of MHC class I chain-related gene B (MICB)
  • Polymerase chain reaction-sequence based typing (PCR-SBT) method and kit of MHC class I chain-related gene B (MICB)
  • Polymerase chain reaction-sequence based typing (PCR-SBT) method and kit of MHC class I chain-related gene B (MICB)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Adopt PCR amplification primer and sequencing primer described in nucleotide sequence table (the position of described primer is as follows figure 1 shown), the high-resolution genotyping of MICB loci was carried out on human blood samples, and the specific steps were as follows:

[0071] (1) Genomic DNA extraction

[0072] Extraction was performed according to the operating instructions of Promaga's Genomic DNA Extraction Kit.

[0073] (2) PCR amplification

[0074] Use MICB site amplification primers MICB-F and MICB-R to amplify MICB typing. The above amplification process and amplification reaction system are as follows:

[0075] The PCR amplification reaction conditions are:

[0076] 1.95℃for 5min

[0077] 2.94°C for 45s → 65°C for 45s (decrease 0.5°C per cycle) → 72°C for 2min (repeat 19 cycles)

[0078] 3.94°C for 45s→55°C for 45s→72°C for 2min (repeat 13 cycles)

[0079] 4.72°C for 10min

[0080] 5.4℃ hold

[0081] The PCR reaction system is 20 μl, and it...

Embodiment 2

[0108] Example 2, the experimental results of the international quality control of UCLA University in the United States and the quality control of the China Bone Marrow Bank

[0109] The HLA quality control samples of the China Bone Marrow Bank are basically selected from the common allele combinations of Chinese people, and the reagent of the present invention has not found any missing detection phenomenon, which is completely consistent with the result of the control reagent. The HLA quality control samples of UCLA University in the United States include the HLA allele combinations of various races around the world, and the results are also consistent with the control reagents.

[0110]

[0111]

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Abstract

The invention provides a polymerase chain reaction-sequence based typing (PCR-SBT) method and a kit of a MHC class I chain-related gene B (MICB). Through the PCR-SBT method and the kit, MICB locus exons 2-5 can be typed. The PCR-SBT method comprises the following steps of 1, preparing a human genome DNA, 2, carrying amplification of target gene fragments needing to be amplified by PCR amplimers, wherein the target gene fragments comprise MICB exons 2-5 and introns between the MICB exons 2-5, and 3, carrying out amplification of the PCR products obtained by the step 2 with sequencing primers, carrying out sequencing of the amplified PCR products, and comparing the sequencing result and a standard sequence in a data base to confirm a gene typing result. Through the optimal combination of test conditions and the kit with MICB sequencing functions, oligonucleotide sequences of whole MICB locus exons 2-5 and a part of introns can be amplified effectively, and sequencing of the corresponding exons is realized. Therefore, through the PCR-SBT method and the kit, accurate MICB typing is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method for amplifying and typing a human MHC class Ichain-related gene B (MICB) site and a matching kit for the method. Background technique [0002] MHC-I chain-related gene (MHC class I chain-related gene, MIC), located in the major histocompatibility complex (MHC) gene region of human chromosome 6, with a length of 2Mb, the encoded protein molecule It is an important ligand recognized by the activating receptor NKG2D of immune cells such as NK cells, γδT cells and CD8+T cells. So far, seven MIC gene loci have been found, and only MICA and MICB are functional genes, which are located at the 46kb and 140kb positions of the centromere, respectively, and are highly linked to the HLA-B locus. The MICB gene has a wide range of polymorphisms, and there are multiple genotypes. According to the sequence differences of exons 2-5 encoding the extracellular region of MICB molecule, 31 M...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 余平龚拯罗奇志林琳霍治杜昆
Owner CENT SOUTH UNIV