FOXP3 (forkhead/winged helix transcription factor 3) gene methylation detection method based on bisulfite sequencing

Abstract

The invention provides an FOXP3 (forkhead / winged helix transcription factor 3) gene methylation detection method based on bisulfite sequencing. The method comprises the following steps: 4 mL of human venous blood is collected by a coagulation-promoting tube, solidified at room temperature for 30 min and centrifuged at 1,000 g*15 min; genomic DNA of a blood clot is extracted; integrity, concentration and purity of the genomic DNA are determined primarily; the genomic DNA is modified with a methylation kit; methylation PCR amplification is performed, a PCR product is identified with 2% agarose gel electrophoresis, and a gel imaging system performs observation and shooting; 18 mu L of the PCR product is purified and recovered by a DNA purifying and recovering kit, 2 mu L of the purified and recovered PCR product is transformed into Trans1-T1 competent cells through vector linking, blue-white clone plaque selection is performed, and positive clone is identified; a kanamycin monoclonal antibody LB liquid medium with the concentration of 50 mu g / mL is inoculated with thalli in positive clone plaques, the thalli are subjected to shake culture at the constant temperature of 37 DEG C and the rate of 200 rpm / min for 8-12 h, and samples turning to be turbid are taken for sequencing. Methylation degrees of 5 CpG sites of a specific sequence of a promoter region of an FOXP3 gene can be detected successfully with the method.

Description

technical field [0001] The invention relates to the biological field, in particular to a method for detecting methylation of the FOXP3 gene based on a bisulfite sequencing method. Background technique [0002] FOXP3 is a characteristic marker of regulatory T cells (regulatory T cells, Treg), and plays an important role in maintaining immune tolerance. Studies in recent years have confirmed that the expression of FOXP3 gene and the function of protein are closely related to epigenetics (such as DNA methylation, post-translational modification, etc.). Bisulfite sequencing is the gold standard for detection of deoxyribonucleic acid (DNA) methylation sites recognized by scholars at home and abroad. It can accurately and sensitively detect different cytosine guanine dinucleotide (CpG) positions in specific DNA sequences. However, it is affected by many factors in the experimental process (such as sample storage conditions, genomic DNA content and purity, genomic DNA content afte...

AI Technical Summary

Problems solved by technology

Bisulfite sequencing is the gold standard for detection of deoxyribonucleic acid (DNA) methylation sites recognized by scholars at home and abroad. It can accurately and sensitively detect different cytosine guanine dinucleotide (CpG) positions in specific DNA sequences. However, it is affected by many factors in the experimental process (such as sample storage conditions, genomic DNA content and purity, genomic DNA content after transformation, PCR reaction system, methylated primer sequences, blue and white clone screening and other experimental factors) low impact success rate

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