Kit and method for detecting STAT3 gene mutation of high IgE (Immunoglobulin E) syndrome
A kit and gene technology, applied in the field of high IgE syndrome STAT3 gene mutation detection kits
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Embodiment 1
[0146] A primer for detecting the polymorphic mutation site of STAT3 gene, the design of the primer is an amplification primer designed for the whole exon of STAT3, including:
[0147] The primers for amplifying the whole exon sequence of STAT3 gene, its base sequence is:
[0148] ST-E1-F: TGTAAAACGACGGCCAGTCTCCGCCCTTTTCTCCTAGC
[0149] ST-E1-R: AACAGCTATGACCATGGCGTCCATCACAACATCCC
[0150] ST-E2-F: TGTAAAACGACGGCCAGT AGCTGTGATTATCCCAAGGTGG
[0151] ST-E2-R: AACAGCTATGACCATG TGCCCAAGTAAATGAGGTCCA
[0152] ST-E3-F: TGTAAAACGACGGCCAGTGTGTATGCGTCGGCTTCA
[0153] ST-E3-R: AACAGCTATGACCATGGACTCTGCGGGTCCTGTT
[0154] ST-E4-F: TGTAAAACGACGGCCAGT AGTAACGACCTCCCCTTCGC
[0155] ST-E4-R: AACAGCTATGACCATGTTGGGTCTGTTGGATTCTTTTGGTG
[0156] ST-E5 / 6-F: TGTAAAACGACGGCCAGT GACCTGTGTAGTGTCACCTGT
[0157] ST-E5 / 6-R: AACAGCTATGACCATG GGACCCGTACCTCCCTTGC
[0158] ST-E7 / 8-F: TGTAAAACGACGGCCAGT TCCCTCAGGTCAAGGAGT
[0159] ST-E7 / 8-R: AACAGCTATGACCATG CTGGCCGAAATAAAGTAAAA
[0160] ST-E9 / 10-F:...
Embodiment 2
[0192] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):
[0193] (1) Extract tissue DNA from blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inverting, and place at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed. 2) Add 20 μl proteinase K solution and mix well. 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap. 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap. 5) Add the solution and flocculent precipitate obtained in the previous step ...
Embodiment 3
[0220] Three cases of clinical samples (sample number 1-3) were taken according to the reagents and methods of Examples 1 and 2 to extract genomes, prepare reagents, amplify and sequence. Add 1 μl of each sample to the detection system PCR reaction solution. Electrophoresis results such as Figure 2-4 Shown, show that the primers ST-E1-F / R, ST-E2-F / R, ST-E3-F / R, ST-E4-F / R, ST-E5 / 6-F / R of the present invention , ST-E7 / 8-F / R, ST-E9 / 10-F / R, ST-E11-F / R, ST-E12 / 13 / 14-F / R, ST-E15 / 16-F / R , ST-E17 / 18-F / R, ST-E19 / 20-F / R, ST-E21-F / R, ST-E22 / 23-F / R, ST-E24-1-F / R, ST -E24-2-F / R, ST-E24-3-F / R, ST-E24-4-F / R can effectively amplify blood samples with a single band.
[0221] Some test results of sample 1 are as follows: Figure 5 , 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19:
[0222] Figure 5 It shows the wild-type sequencing screenshot of STAT3 exon 1 of sample 1,
[0223] Figure 6 It shows the wild-type sequencing screenshot of STAT3 exon 2 of sample 1,
[0224] Figur...
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