Human SDC2 gene methylation detection kit

A detection kit and methylation technology, applied in the field of human SDC2 gene methylation detection kits, can solve the problems of cumbersome operation, increased difficulty, incomplete transformation, etc., and achieve fast and simple operation, easy automation, and guaranteed accuracy. Effect

Active Publication Date: 2020-01-17
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bisulfite conversion method has the following disadvantages: ①The conversion conditions are relatively severe, which may cause DNA degradation and affect the sensitivity of subsequent detection; ②The operation is cumbersome, and purification is required after conversion, which may cause DNA loss; ④ Unmethylated cytosine accounts for 95% of the total cytosine in the human genome, and the complete conversion of unmethylated cytosine to thymine will seriously reduce the sequence complexity, thereby Reduces sequence specificity and increases the difficulty of subsequent specificity detection
[0009] In addition, various methylated SDC2 detection methods developed based on bisulfite conversion also have their own shortcomings
For example, the bisulfite sequencing method requires a large number of clones to be sequenced, which is cumbersome and expensive; the sequence length of each sequence of pyrosequencing is limited (Grigg G et al., Bioessays, 2010, 16, 431-436); A Methylation-specific PCR and real-time methylation-specific PCR lack effective mechanisms to prevent non-specific amplification

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  • Human SDC2 gene methylation detection kit
  • Human SDC2 gene methylation detection kit
  • Human SDC2 gene methylation detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Preparation of the human SDC2 gene methylation detection kit (fluorescent PCR method)

[0045] Human SDC2 gene methylation detection kit (fluorescence PCR method), including enzyme digestion / fluorescence PCR reaction system, negative quality control and positive quality control. The preparation of the kit includes the following steps:

[0046] (1) Design and preparation of labeled twin primers: for the CpG island of the SDC2 gene, the region of the ACTB gene containing the cleavage site of the methylation-sensitive restriction endonuclease, and the region of the ACTB gene that does not contain the methylation-sensitive restriction endonuclease For the region of the restriction site of Dicer, the labeled twin primers for SDC2 gene methylation detection, the enzyme digestion internal reference labeled twin primers, and the PCR internal reference labeled twin primers were respectively designed, as shown in SEQ ID 1 to SEQ ID 12. Primers were stored in 100 μM...

Embodiment 2

[0063] Example 2: Comparative experiment of using different methylation sensitive restriction endonucleases to detect SDC2 methylation

[0064] (1) Purpose of the experiment

[0065] In this example, different methylation-sensitive restriction endonucleases were used to detect the methylation of SDC2 to compare the digestion effects of different methylation-sensitive restriction endonucleases.

[0066] (2) Experimental method

[0067] In this example, according to the preparation steps of the kit in Example 1, AciI, HinP1I, and HpaII were respectively used as methylation-sensitive restriction enzymes to prepare three kinds of restriction endonuclease / fluorescent PCR reaction systems; using the above three enzymes Enzyme cutting / fluorescent PCR reaction system Respectively perform enzyme cutting / fluorescent PCR reactions on three copies of SDC2 gene unmethylated human genomic DNA and SDC2 gene methylated human genomic DNA at the same concentration. Other components of the kit...

Embodiment 3

[0073] Example 3: Verification experiment of detection effect of labeled twin primers

[0074] (1) Purpose of the experiment

[0075] In this example, the detection effect of the labeled twin primers of the present invention is verified by comparing with the detection results of traditional primers.

[0076] (2) Experimental method

[0077] In this embodiment, methylated human genomic DNA is selected as the sample to be tested. Use the kit described in Example 1 prepared by labeled twin primers and traditional primers (traditional primers are single-stranded primers, the base sequence of which is the same as that of the upstream and downstream forward primers of labeled twin primers, and its 5' end without fluorescence Group) combined with fluorescent probes to establish an enzyme digestion / fluorescent PCR reaction system to detect methylated human genomic DNA of the SDC2 gene in three copies of the same concentration. At the same time, use the SDC2 labeled twin primers des...

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Abstract

The invention provides a human SDC2 gene methylation detection kit, which comprises an enzyme digestion reaction solution and a fluorescent PCR (polymerase chain reaction) solution, wherein the enzymedigestion reaction solution and the fluorescent PCR reaction solution are packaged in a same PCR amplification tube in layered form through a hot melting material, and the enzyme digestion reaction solution occurs at the bottom of the PCR amplification tube. According to the kit disclosed by the invention, methylation sensitive restriction endonuclease and marking twin primers are utilized to carry out enzyme digestion reaction and fluorescent PCR sequentially in one tube so as to detect the methylation state of the SDC2 gene; the kit has the advantages of high operation speed, good operationsimplicity, high sensitivity, good specificity and good convenience of automation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human SDC2 gene methylation detection kit. [0002] technical background [0003] Syndecan-2 (Syndecan-2, SDC2) is a transmembrane heparan sulfate proteoglycan that regulates many cellular functions critical to tissue development and homeostasis, including cell proliferation, differentiation, adhesion , cytoskeleton organization, migration, wound healing, angiogenesis, etc. (Mytilinaiou M et al., IUBMB Life, 2017, 69, 824-833). The SDC2 gene encoding this protein contains 9 exons and is located on human chromosome 8q22.1. SDC2 is associated with many different types of tumors, and changes in its expression have been detected in a variety of tumors (Mytilinaiou M et al., IUBMB Life, 2017, 69, 824-833). In human colon adenocarcinoma tissue samples, the mRNA and protein expression of SDC2 was increased compared with adjacent normal epithelium, and this upregulation was ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2521/301C12Q2531/113C12Q2563/107C12Q2535/101C12Q2547/101Y02A50/30
Inventor 许嘉森吴诗扬彭璨璨刘志明刘芳
Owner SUREXAM BIO TECH
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