CDH23 gene mutant, kit for detecting CDH23 gene mutant and application of kit
A technology of kits and reagents, applied in the field of molecular biology, to achieve the effect of high early diagnosis rate
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Embodiment 1
[0029] In the CDH23 gene mutant of this embodiment, compared with the nucleotide sequence of the wild-type CDH23 gene, the nucleotides of the CDH23 gene mutant have the following mutations: c.3890G>T and / or c.6649A>G; The wild-type CDH23 gene has the nucleotide sequence of SEQ.ID.No.5 in the sequence table; the above missense mutation c.3890G>T is a mutation from G to T relative to the 3890th position of the cDNA of the wild-type CDH23 gene , c.6649A>G is a mutation from A to G relative to the 6649th position of the cDNA of the wild-type CDH23 gene.
[0030] In this example, a new mutant of the CDH23 gene is identified, which is closely related to the onset of sensorineural deafness, so by detecting whether the new mutant exists in a biological sample, it can effectively detect whether the biological sample is susceptible to Sensorineural deafness.
[0031] The nucleic acid encoding the CDH23 mutant is detected and verified by the method of whole exome sequencing, family anal...
Embodiment 2
[0052] This example is to determine the pathogenic gene of sensorineural deafness.
[0053] The genes of a 2-generation Chinese family with sensorineural deafness and 100 normal individuals outside the family were collected. figure 1 Pedigree maps of families with patients with sensorineural deafness are shown. Among them, □ indicates a normal male, ○ indicates a normal female, and ● indicates a female patient. There are 5 members in this family, of which the two daughters of the second generation are patients with sensorineural deafness, and the parents of the first generation are normal members.
[0054] DNA extraction
[0055] The peripheral venous blood of 2 patients in the family, 2 normal people in the family and 100 normal people outside the family were collected respectively, and the genomic DNA of the peripheral blood leukocytes was extracted by QIAmp Bloodkit (Qiagen, Hilden, Germany), and the DNA was extracted by QubitFluorometer and agarose Gel electrophoresis t...
Embodiment 3
[0061] The pathogenic genes of sensorineural deafness were verified by Sanger sequencing.
[0062] Respectively to 2 patients in embodiment 2 ( figure 1 Middle Ⅱ:1, Ⅱ:2), 2 normal people in the family ( figure 1 Ⅰ:1, Ⅰ:2) and 100 normal human genes outside the family were tested, and primers were designed for the sequence of the compound heterozygous mutation site of the CDH23 gene, and relevant sequences were obtained by PCR amplification, product purification, and sequencing. The correlation was verified according to whether the sequence determination results belonged to the mutant type or the wild type, and whether the mutation and the phenotype co-segregated in the family. The specific method steps are as follows:
[0063] 1) DNA extraction:
[0064] The peripheral venous blood of 2 patients in the family, 2 normal people in the family and 100 normal people outside the family were used to extract genomic DNA, and the DNA content and purity were determined.
[0065] 2) ...
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