Human SDC2 gene methylation detection kit

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...

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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