Detection probe for DUOX2 gene mutation and detection method thereof
A detection method and gene technology, applied in the field of molecular biology, can solve the problems of high cost, limited clinical application, and long manual operation time.
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Embodiment 1
[0125] Embodiment 1: CH polygenic PCR primer design
[0126] Use oligo software to design DUOX2 gene PCR amplification primers. The primer design follows the following principles: the length of the primer is suitable for 19-23bp; the primer should have no dimer, especially the possibility of dimer formation at the 3' end; Hairpin structure; the difference between upstream and downstream primer Tm values should not exceed 5°C; GC content should be 45-55%. After the upstream and downstream primers are determined, use Blast to compare and analyze the upstream and downstream primers in the NCBI database to ensure the specificity and amplification efficiency of the primers. According to the distance between the exons, some exons were combined to design primers. A total of 9 pairs of primers were designed for the 33 exons, which were synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. The upstream primers P-F of the above exons and the downstream primers The sequence o...
Embodiment 2
[0145] Example 2: Collection of blood samples and extraction of genomic DNA:
[0146] A total of 192 unrelated CH patients from Guangxi (age: 9 days to 6 years old, median age 18 days) were selected according to the diagnostic criteria of the "Technical Standards for Newborn Disease Screening" issued by the Ministry of Health. All subjects or family members signed a written informed consent form. This study was approved by the Ethics Committee of our hospital and complied with the World Medical Association Declaration of Helsinki: Ethical Principles for Human Medical Research.
[0147] 192 cases of blood samples were prepared according to the following method, using the kit: Tiangen Biochemical Technology (Beijing) Co., Ltd., Blood Genome Extraction Kit (DP318)
[0148] 1. Add 1000 μl of ligsis buffer to 500 μl of anticoagulated blood, and shake thoroughly until clear. Centrifuge at 4000rpm for 5min. Discard the supernatant.
[0149] 2. Add 1500 μl of ligsis buffer to the p...
Embodiment 3
[0158] Example 3: Target Region Amplification
[0159] All primers were sent to Shanghai Sangong for synthesis. After the primers were synthesized, they were centrifuged at 12,000g for 2 minutes to precipitate the dry powder. According to the instructions, add high-pressure sterilized double-distilled water to dilute to a final concentration of 10uM. After the primers were fully dissolved, they were used in PCR experiments. The annealing temperature of all primers was explored using the American AB Veriti gradient PCR instrument, and the PCR amplification system was 25 μl. For target fragments with high GC content, DMSO was added to the system to a final concentration of 2%. For primers with low specificity for the amplified bands, it is usually considered to change the annealing temperature or redesign the primers to ensure that the amplified target bands are clear and specific.
[0160] 1. Primer sequence: it is the nucleotide sequence of SEQ ID No.1-SEQ ID No.18 in the seq...
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