A method and primers for detecting all exons of an IL7R gene
An all-exon, sequencing primer technology, used in life sciences and biology, can solve problems such as weakening the effect of steroid hormone therapy
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Embodiment 1
[0091] A primer for detecting the polymorphic mutation site of IL7R gene, the design of the primer is the amplification primer designed for the whole exon of IL7R, including:
[0092] The primers for amplifying the whole exon sequence of IL7R gene, its base sequence is:
[0093] IL7R-Exon1-F TGTAAAACGACGGCCAGTGGCTCTGTCTCTTCATGTCCTTG
[0094] IL7R-Exon1-R AACAGCTATGACCATGTGACTCAGGTCTGTTAGGGAACTG
[0095] IL7R-Exon2-F TGTAAAACGACGGCCAGTGTCTGCCACAGAGTCTGCTATT
[0096] IL7R-Exon2-R AACAGCTATGACCATGGCTGAACCGTGAGAGGAGATT
[0097] IL7R-Exon3-F TGTAAAACGACGGCCAGTTTCCTGAACATGCCTCCACTC
[0098] IL7R-Exon3-R AACAGCTATGACCATGTAACCTACAGAATGTCCAGACACA
[0099] IL7R-Exon4-F TGTAAAACGACGGCCAGTCTGGAGAATGCGGACTGGAT
[0100] IL7R-Exon4-R AACAGCTATGACCATGTGCCTGATTTGCCTATGAAAGTG
[0101] IL7R-Exon5-F TGTAAAACGACGGCCAGTCACTCTCCCTCCTTGACCACTC
[0102] IL7R-Exon5-R AACAGCTATGACCATGCTCTCACTTGCTCCCACACTT
[0103] IL7R-Exon6-F TGTAAAACGACGGCCAGTCAAAGCACCCTGAGACCCTAC
[0104]IL7R-Exon6-R AACAGCT...
Embodiment 2
[0119] Bone Marrow / Blood / Tissue Genomic DNA Extraction Kit (Tiangen Biological) operation process:
[0120] (1) Extract tissue DNA from blood: 1) Extract 300 μL of bone marrow or blood and add 900 μL of erythrocyte lysate, mix by inversion, leave at room temperature for 5 minutes, and mix by inversion several times during the period. 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 solution GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap. 4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, and centrifuge briefly to remove the water droplets in the tube cap. 5) Add the solution and flocculent precipitate obtained ...
Embodiment 3
[0159] Take 16 cases of clinical samples (numbered ex1-ex8 according to the primers), and extract genomic DNA, prepare reagents, amplify and sequence according to the reagents and methods of Examples 1 and 2. Add 1 μL of the sample to the detection system PCR reaction solution. Electrophoresis results such as Figure 1-a to Figure 1-h As shown, it shows that the primers of the present invention can effectively amplify blood samples, and the band is single.
[0160] The partial sequencing results of the samples are as follows: figure 2 Shown: (a) shows the wild-type sequencing screenshot of exon 5 of IL7R gene in sample No. 1, indicating that no mutation occurs in exon 5 of IL7R in the sample; Screenshot of the wild-type sequencing of the offspring, indicating that the IL7R exon 6 of the sample is not mutated;
[0161]It can be seen from the detection results that the primers of the present invention have included exon sequences, can amplify all exons of the IL7R gene, and ...
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