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A method and kit for detecting SOCS family gene tag single nucleotide polymorphism site

A polymorphic site, single nucleotide technology, applied in the field of molecular biology, can solve problems such as genetic association research between limited gene polymorphisms and disease occurrence, development and prognosis, and achieve economic detection, accurate detection and interpretation. , the effect of high efficiency

Inactive Publication Date: 2016-04-20
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more than 20 kinds of SNP detection methods have been developed, such as direct sequencing, probe hybridization, flight mass spectrometry, restriction fragment length polymorphism analysis, etc., but due to the cost, accuracy and applicable scale of these methods, etc. The disadvantages in this field limit the research on the genetic association between gene polymorphisms and disease occurrence, development and prognosis

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  • A method and kit for detecting SOCS family gene tag single nucleotide polymorphism site
  • A method and kit for detecting SOCS family gene tag single nucleotide polymorphism site
  • A method and kit for detecting SOCS family gene tag single nucleotide polymorphism site

Examples

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Effect test

Embodiment 1

[0035] Example 1 Screening of Candidate Gene Tag Single Nucleotide Polymorphism Sites

[0036] Use the HapMap (http: / / www.hapmap.org) SNP database (HapMapDataRel28PhaseII+III, Aug10, onNCBIB36assembly, dbSNPb126) to download each gene (CIS, SOCS2-7) and its upstream and downstream 3kb within the Chinese Beijing Han population (CHB) SNP data: 1) Enter the gene name in the search box to view the location of each gene in HapMap, and each extend 3kb upstream and downstream; 2) Select from the "Reports&Analysis" option: DownloadSNPgenotypedata. 3) Select Chinese Han Beijing (CHB) in "config" to obtain all SNP sites in the SOCS family genes and their extended regions of the Chinese Beijing Han population.

[0037] Open the program running window under the TAGster software package directory. According to the criteria for constructing bins and selecting label SNPs (minimum allele frequency ≥ 0.05 and linkage relationship between SNPs r 2 ≥0.8) Set the parameters and run the commands...

Embodiment 2

[0042] Example 2 Design and Synthesis of Single Nucleotide Polymorphism Site PCR Amplification Primers and Pyrosequencing Primers

[0043] Using NCBI ( www.ncbi.nlm.nih.gov )GenBank downloads about 500bp genome sequence of each tag SNP site and its vicinity. Use the software "AssayDesign" attached to the pyrosequencer to design PCR amplification primers for single nucleotide polymorphism sites and pyrosequencing primers, and select the primer pair with the highest score. The designed primers were synthesized by Shanghai Bioengineering Co., Ltd., and one of the PCR primers was modified with biotin at the 5' end. The designed 12 tag SNP site amplification primers and sequencing primers are shown in Table 2.

[0044] Table 212 Index SNP site amplification primers and sequencing primers

[0045]

Embodiment 3

[0046] Example 3 Whole Blood Genomic DNA Preparation

[0047] Check the whole blood genomic DNA purification kit (take the reagents produced by Promega in the United States as an example), the shelf life of pyrosequencing reagents and ensure that ethanol has been added to the WashBuffer and mark the bottle cap; (2) isopropanol and 75% ethanol; (3) 1.5ml Eppendorf (EP) tubes and various types of pipettes and tips within the validity period of autoclaving.

[0048] Take out the peripheral blood of trauma patients collected in EDTA anticoagulant tubes from the -80 refrigerator, thaw at room temperature and mix upside down several times; pipette 300ul whole blood and 900ul CellLysisSolution into 1.5ml EP tubes marked with corresponding samples, and incubate at room temperature for 10 minutes , mix once every 2-3 minutes; centrifuge at 10000g, 4°C for 1 minute; take out the EP tube, observe the white precipitate, and discard the red supernatant; repeat the operation 2-3 times until...

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Abstract

The invention belongs to the filed of molecular biology, and specially relates to a kit and a method for detecting SOCS family gene labeled single nucleotide polymorphism sites, which are applicable to single and double PCR combined pyrosequencing technologies. The method specially comprises the following steps: according to a confirmed SOCS family gene labeled single nucleotide polymorphism site, designing specific amplification primers and sequencing primers, wherein one of the specific amplification primers is used for labeling a biotin; and through taking the DNA of a specimen to be detected as a template, carrying out PCR reaction by using the specific amplification primers so as to obtain a PCR amplification content; dividing the obtained PCR amplification content into single strands by using alkaline denaturation, after a biotin labeled single strand PCR amplification product is taken for purification, carrying out hybridization reaction on the obtained product and the sequencing primers, and analyzing results. Due to an SOCS target sequence selected in the invention, and through the application of the kit disclosed by the invention, the rapid, simple, accurate, efficient, practical and economical detection of SOCS family gene labeled single nucleotide polymorphic genotypes can be realized, and the needs on the clinical inspection of practical work can be met, thereby facilitating the prediction of the occurrence and development risks of diseases through SOCS gene detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a kit and a method suitable for detecting SNP sites of SOCS family gene tags by combining single and double PCR with pyrosequencing technology. Background technique [0002] Suppressor of cytokine signaling (SOCS), as a negative regulator of cytokine signal transduction expressed in cells, initiates related signal transduction pathways through specific cell surface receptors in cytokines, mediates the development and differentiation of immune cells, and naturally play an important role in the adaptive immune response. However, the excessive release or inhibition of cytokines caused by abnormal regulation of cytokine inhibitory factors may cause various diseases such as allergy, autoimmune disease, inflammation or tumor. Therefore, the changes of SOCS molecules in the occurrence of various diseases are closely related to the occurrence, development and prognosis of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 张安强蒋建新顾玮岳彩黎王海燕杜鹃
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA