SFRP2 methylation detection kit and application thereof
A detection kit and methylation technology, applied in the field of SFRP2 methylation detection kits, can solve the problems of reducing sample complexity, small number of DNA samples, PCR amplification failure, etc. False positive problem, the effect of improving detection specificity
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Embodiment 1
[0074] Example 1 Detection of methylated DNA of human SFRP2 gene
[0075] In this example, methyltransferase-treated Jurkat DNA was used as a positive standard for methylation of human SFRP2 gene, and nuclease-free water was used as a negative control, wherein the CG site was 5mCG, and the methylation site of SFRP2 gene was detected. Proceed as follows:
[0076] (1) Methylation-dependent restriction endonuclease GlaI was used to digest the positive standard and negative control. The reaction system was 2 μL of 10× digestion buffer, 5U GlaI, and different concentrations of genomic DNA, and the total system was 20 μL ; The reaction conditions were incubated at 30°C for 1 hour; after the enzyme cleavage reaction, the system was heated to 85°C and incubated for 10 minutes to heat inactivate GlaI;
[0077] (2) Add SFRP2 gene capture oligonucleotides, universal primers, specific primers and detection probes to the above enzyme digestion reaction system respectively, and PCR detects...
Embodiment 2
[0089] Example 2 Amplification results of 2'-Fluoro RNA modification in the folded region of captured oligonucleotides
[0090]Due to the limitations of the existing oligonucleotide synthesis technology, the capture oligonucleotide may produce by-products with incomplete sequence fragments during the synthesis process, which may cause non-specificity in the high concentration of non-methylated background. Amplification, in this example, by modifying the 2'-Fluoro RNA in the folded region of the capture oligonucleotide, the non-specific amplification caused by the residual fragments is effectively suppressed, and the detection specificity is improved.
[0091] The positive templates, universal primers, specific primers, detection probes and reaction conditions used in this example are the same as in Example 1, and the capture oligonucleotides used include:
[0092] 2'-Fluoro RNA modified capture oligonucleotide (SEQ ID NO:6):
[0093] 5-TGTCAGCCAACGGTATTCATCTTTGCGACCC CGAGGG...
Embodiment 3
[0095] Example 3 Methylation detection of human SFRP2 gene in stool samples
[0096] In this example, the methyltransferase-treated Jurkat DNA was used as the positive standard for methylation of the human SFRP2 gene, and the CG site was 5mCG, which was incorporated into the DNA of the stool sample to detect the methylation site of the SFRP2 gene. Nuclease-free water was used as a no-template control. Proceed as follows:
[0097] (1) Methylation-dependent restriction endonuclease GlaI was used to digest the mixed sample and no-template control. The reaction system was 1× PCR buffer (purchased from Nanjing Novizan, P122-d1), 5U GlaI 10 μL of genomic DNA with different concentrations; the reaction conditions were incubation at 37°C for 1 hour; after the digestion reaction, the system was heated to 85°C and incubated for 10 minutes to heat inactivate GlaI;
[0098] (2) Add SFRP2 gene capture oligonucleotide (SEQ ID NO:6), universal primer (SEQ ID NO:2), specific primer (SEQ ID ...
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