or2w3 gene mutant and its application
A technology for amino acids and degenerative diseases, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve problems that need to be further studied
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Embodiment 1
[0062] Example 1 Mutation screening of known sites
[0063] In order to find the causative mutation gene and exclude known genes, the inventors conducted mutation screening on the affected members of the family. In RP patients (II-1) and healthy controls (II-8), peripheral venous blood was collected, and DNA was extracted from peripheral granulocytes using the TIANamp blood DNA extraction kit. The exons of the gene and the splice sites of the flanking introns were sequenced and further confirmed by Sanger sequencing. Previous research results have shown that the genes related to RP are: RP1, RPGR, RP2, RHO, PRPH2, ROM1 / PRPH2, RP9, IMPDH1, PRPF31, CRB1, PRPF8, TULP1, CA4, PRPF3, ABCA4, RPE65, EYS, CERKL , NRL, FAM161A, FSCN2, TOPORS, SNRNP200, SEMA4A, PRCD, NR2E3, MERTK, USH2A, PDE6B, PROM1, KLHL7, PDE6A, RGR, CNGB1, IDH3B, SAG, GUCA1B, CNGA1, BEST1, TTC8, RDH12, C2orf71, ARL6 , IMPG2, PDE6G, ZNF513, DHDDS, PRPF6, CLRN1, MAK, CDHR1, RBP3, RPGR, CC2D2A, FLVCR1, TTPA, OFD1, RLB...
Embodiment 2
[0064] Example 2 Using Whole Exome Sequencing to Identify Pathogenic Genes
[0065] Select 5 members of the family, including 4 RP patients (II-2, II-3, II-4, II-7) and 1 normal member (II-9). 30 μg of human genomic DNA was extracted from the peripheral venous blood of each participant, and used for subsequent experiments after passing the test. According to the operating instructions of the Covaris Acoustic system, the genomic DNA was broken into random fragments, and then the fragments were ligated using adapters to prepare a hybrid library. Human genomic DNA was subjected to ligation-mediated PCR (LM-PCR) amplification, purification, and Nimblegen SeqCap EZ laboratory v3.0 (Roche / NimbleGen, Madison, WI) hybridization enhancement, LM-PCR capture and uncaptured product utilization The degree of enhancement was assessed by Q-PCR. Each captured library was loaded on a Hiseq2500 plate (Illumina, San Diego, CA), and high-throughput sequencing of each captured gene library was p...
Embodiment 3d
[0077] Example 3 Sanger sequencing verification of de novo SNP sites in the family and in the normal population control group
[0078] To determine whether any remaining variants in this family cosegregated with the disease phenotype, the inventors performed Sanger sequencing on DNA samples from other members of the family to confirm the mutation. The direct PCR product was sequenced with ABI3730 Genetic Analyzer, and the obtained data was compared with the human genome database (GRCh37, UCSChg19) to detect mutations. The possible pathogenic mutations were further verified using the RP family tree database of Shenzhen Huada Gene Research Institute, which contains the whole exome sequence data of several RP families.
[0079] Using the peripheral venule blood of each participant as a sample, 30 μg of human genomic DNA was extracted using the TIANamp Blood DNA Extraction Kit, which was used for subsequent experiments after passing the test.
[0080] PCR reaction primer design r...
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