Method and primer for detecting hereditary blood coagulation factor XI(F11) genes
A technology of coagulation factor, F11E1R, applied in the field of medical detection, can solve problems such as easy bleeding
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Embodiment 1
[0127] A primer for detecting the polymorphic mutation site of coagulation factor XI (F11) gene. The design of the primer is an amplification primer designed for the whole exon of coagulation factor XI (F11), including:
[0128] The primers for amplifying the whole exon sequence of coagulation factor XI (F11) gene have the base sequence:
[0129] F11 E1 F: TGTAAAACGACGGCCAGTAGGCACACAGGCAAAATC
[0130] F11 E1 R: AACAGCTTGACCATGGATATTAGCGGAACATCTCTAC
[0131] F11 E2 F: TGTAAAACGACGGCCAGTACACAGTCACACTAAGGAAT
[0132] F11 E2 R: AACAGCTATAGACCATGCTCTCCCAGCCCATAGTT
[0133] F11 E3 F: TGTAAAACGACGGCCAGTGCTACTTGCCTTGCCTT
[0134] F11 E3 R: AACAGCTATAGACCATGGTTAAGAATAGCAATCTCCC
[0135] F11 E4 F: TGTAAAACGACGGCCAGTGCTTTCTGTGTGCTGACT
[0136] F11 E4 R: AACAGCTTGACCATGCGATGTGGCGATATGTGTA
[0137] F11 E5 F: TGTAAAACGACGGCCAGTCTAGAATCTGGAAGGTACTCAT
[0138] F11 E5 R: AACAGCTATAGACCATGGGCATAAAGTTGATGGCAAA
[0139] F11 E6 F: TGTAAAACGACGGCCAGTCAGGTCCTCTCTCCAAAG
[0140] F11 E6 R: AACAGCTTGACCATGGCTGGTATCCTGAG...
Embodiment 2
[0168] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Bio):
[0169] (1) Extract tissue DNA from blood: 1) Draw 300 μl of blood and add 900 μl of red blood cell lysate, mix upside down, place it at room temperature for 5 minutes, and mix upside down several times during the process. Centrifuge at 12,000 rpm for 1 min, aspirate the supernatant, leave the white blood cell pellet, add 200 μl of buffer GA, and shake until thoroughly mixed. 2) Add 20μl proteinase K solution and mix well. 3) Add 200μl of buffer GB, fully invert and mix well, place at 70°C for 10 minutes, the solution should be clear, and centrifuge briefly to remove the water droplets on the inner wall of the cap. 4) Add 200μl of absolute ethanol, shake and mix well for 15 seconds. At this time, flocculent precipitation may appear. Centrifuge briefly to remove water droplets on the inner wall of the cap. 5) Add the solution and flocculent precipitate obtained in the previous step to an ads...
Embodiment 3
[0224] In order to verify the feasibility of the primers and the entire detection system, 15 clinical blood samples (numbered 1-15) were taken. Follow the reagents and methods of Examples 1 and 2 to extract whole genome DNA, prepare reagents, amplify and sequence. Add 1μl of the sample to the PCR reaction solution of the detection system. The results of electrophoresis are as figure 2 It is shown that the primers of the present invention can effectively amplify blood samples and have a single band.
[0225] Sample Exon (1-15) amplification product Sequencing results Point mutation Extra gene insertion Ploidy 1 Clear and single band Single peak without clutter no no no 2 Clear and single band Single peak without clutter no no no 3 Clear and single band Single peak without clutter no no no 4 Clear and single band Single peak without clutter no no no 5 Clear and single band Single peak without clutter no no no 6 Clear and single band Single peak without clutter ...
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