Application of SNP bite of CLIP3 gene
A site, aortic dissection technology, applied in the field of biomedical detection, can solve the problems of high AD vascular risk, difficult early risk assessment, and difficult early detection
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Embodiment 1
[0029] Example 1 Sample collection
[0030] From January 2017 to April 2018, 59 patients with Stanford type A aortic dissection were diagnosed by aortic CTA in Shenzhen Sun Yat-sen Cardiovascular Hospital, and 2 mL of whole blood and 590 normal control samples were collected from the searched by Makino Sequencing Company. database. Informed consent was obtained from the patient and reviewed by the Ethics Committee.
[0031] Sample processing: EDTA anticoagulated whole blood was mixed with Trizol at a ratio of 1:1, mixed thoroughly and placed in a 1.8mL cell cryopreservation tube, cooled rapidly in liquid nitrogen for 30 seconds and stored in a -80°C refrigerator.
Embodiment 2
[0032] Embodiment 2 extracts blood sample DNA
[0033] (1) Add 1 mL of CL cell lysate to 1 mL of EDTA (0.01M, China, Huamei Bioengineer) anticoagulated blood, gently invert and mix 6 times, centrifuge at 3600 rpm for 5 min, and discard the supernatant;
[0034] (2) Pour 1mL of CL cell lysate into the centrifuge tube again, gently invert and mix 6 times, centrifuge at 3600rpm for 5min, discard the supernatant; under the premise of ensuring that the precipitate remains in the tube, invert the centrifuge tube Stand on clean absorbent paper for 2 minutes;
[0035] (3) configuring a mixed solution of proteinase K and buffer FG;
[0036] (4) Add 500 μL of proteinase K and buffer FG mixture, then mix until the solution has no lumps;
[0037] (5) Water bath at 65°C for 30 minutes, and mix by inverting several times during the period;
[0038] (6) Add 1 mL of isopropanol, then invert and mix until clustered or filamentous genomic DNA appears;
[0039] (7) Centrifuge at a speed of 3...
Embodiment 3
[0047] Example 3 Whole Exome Sequencing
[0048] 1. DNA library construction
[0049] 3 μg of DNA was ultrasonically fragmented, the ends of the interrupted fragments were blunted, A was added to the 3' end, and the adapter was connected, and the fragments between 350 and 400 bp were selected to prepare the genome-wide library. Library samples were quality controlled using an Agilent 2100 bioanalyzer (Agilent Technologies, USA).
[0050] 2. Target region capture sequencing
[0051] Whole-exome detection was performed using GenCap liquid-phase capture target gene technology (Beijing Mikino Company). Mix 1 μg of DNA library with BL buffer and probe, heat at 95°C for 7 minutes, heat at 65°C for 2 minutes, add 23 μL of HY buffer preheated to 65°C, and hybridize at 65°C for 22 hours. 50 μL MyOne magnetic beads (Life Technology, USA) were washed 3 times with 500 μL 1X binding buffer, and resuspended in 80 μL 1X binding buffer. Add 64 μL 2X Binding Buffer to the hybridization mix...
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