Detection primer and detection system for methylation of SNRPN (Small Nuclear Ribonucleoprotein-associated Protein N) gene promoter region
A technology for gene promoter regions and detection primers, which is applied in DNA/RNA fragments, microbial determination/inspection, biochemical equipment and methods, etc. It can solve problems affecting the accuracy and reliability of experimental results, increase experimental steps and experimental time and other problems, to achieve the effect of simple data analysis method, reduced detection cost, and good specificity
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Embodiment 1
[0035] An embodiment of the SNRPN gene promoter region methylation detection kit of the present invention mainly includes the following components: (1) detection primers, which include forward primers and reverse primers, wherein the forward primer nucleoside The acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; (2) DNA extraction reagent; (3) DNA sulfite conversion reagent; (4) HRM PCR reaction Reagents; (4) Quality control: negative quality control (NEG), positive quality control A (AS), positive quality control B (PW).
[0036] The detection primers are synthesized in a conventional way, prepared as a mother solution according to the instructions, and ddH can be used to 2 O was diluted to a working concentration of 5uM.
[0037] The DNA extraction reagent was purchased from QIAGEN, Germany, with the item number 51106.
[0038] The DNA sulfite conversion reagent was purchased from ZYMO Research Company, the pr...
Embodiment 2
[0041] Such as figure 1 As shown, this embodiment provides a detection system 10 for the methylation of the promoter region of the SNRPN gene, including an extraction module 100, a pre-processing module 110, a reaction module 120 and an analysis module 130.
[0042] The extraction module 100 is used to extract the DNA of the sample to be tested;
[0043] The pretreatment module 110 is used to convert the DNA obtained by the extraction module into sulfite;
[0044]The reaction module 120 utilizes the detection primers to detect the methylation ratio of the SNRPN gene promoter region on the DNA obtained by the pretreatment module;
[0045] The analysis module 130 is used to analyze and process the information obtained by the reaction module to obtain detection results.
Embodiment 3
[0047] A kind of embodiment of detection method of methylation of SNRPN gene promoter region of the present invention, utilizes the kit described in embodiment 1 and the detection system described in embodiment 2 to detect, comprises the steps:
[0048] (1) adding the patient's blood sample into the extraction module 100 to extract DNA;
[0049] (2) Take 200ng DNA into the pretreatment module 110 for sulfite conversion, and convert cytosine to uracil: A. Prepare CT conversion reagent as follows: add 900 μl water, 300 μl M-DilutionBuffer and 50 μl M-Dissolving Transfer the Buffer to the CT conversion reagent tube, use a rocking mixer to shake at room temperature for 10 minutes, then prepare the reaction system as described in Table 1, and perform the reaction according to the procedures described in Table 2; B. Add 600 μl M-Binding Buffer to Zymo-Spin TM IC Column, then add the transformed DNA to the column, blow and mix twice; C, centrifuge at full speed for 30s; change a new ...
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