crx gene mutant and its application
A technology of mutants and constructs, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of cone and rod cells that need to be deepened, and the etiology of patients is unknown, so as to achieve the effect of high early diagnosis rate
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Embodiment 1
[0068] Example 1 Determining the Causative Mutation of Cone and Rod Dystrophy
[0069] 1. Sample collection
[0070] The inventor has an adCORD diseased family living in southern China, the pedigree is shown in figure 2 . Such as figure 2 As shown, ○ indicates normal female, □ indicates normal male, ■ indicates male patient, ● indicates female patient, Indicates a deceased male patient; Indicates a deceased normal male; Indicates a deceased normal female; the arrow points to the proband (III-12).
[0071] There are 9 patients in this family, with 3 consecutive generations of the disease, both men and women are affected, which conforms to the Mendelian autosomal dominant inheritance pattern. The proband (III-12) of this family was male, aged 50. The proband was diagnosed with progressive vision loss and slight color vision defect at the age of 40. Fundus photography showed a 2×2PD gold leaf image in the patient's macula with diffuse hypopigmentation; the peripheral ...
Embodiment 2
[0089] Example 2 Sanger method sequencing verification
[0090] Detect the CRX gene of all family members (including patients and normal family members) in the adCORD patient family described in Example 1, and 100 normal people outside the family line: design primers for the c.766C>T mutation of the CRX gene , and then obtain the relevant sequence of the mutation site by PCR amplification, product purification and sequencing, and verify the relationship between the CRX gene and the mutation and cone-rod cell dystrophy according to whether the sequence determination result belongs to the mutant type or the wild type Correlation.
[0091] The specific method steps are as follows:
[0092] 1. DNA extraction
[0093] According to the method for extracting DNA described in Example 1, the genomic DNA in the peripheral venous blood of the subject was extracted and prepared respectively for future use.
[0094] 2. Primer design and PCR reaction
[0095] First, with reference to th...
Embodiment 3
[0108] Example 3 Detection Kit
[0109] Preparation of a detection kit comprising primers capable of detecting the c.766C>T mutation of the CRX gene for screening biological samples susceptible to cone-rod dystrophy, wherein the primers are specific to the exons of the CRX gene Primers, the sequences of which are shown in SEQ ID NO: 3-4 in Example 2.
[0110] The specific steps for using the above kit to screen biological samples susceptible to cone-rod dystrophy are: extract the DNA of the test subject according to the method described in 2.1 "DNA Extraction" of Step 2 of Example 1, and use the extracted DNA is used as a template to perform a PCR reaction with the exon-specific primers of the above-mentioned CRX gene (see Example 2 for the PCR reaction system and reaction conditions), and the PCR product is purified according to a conventional method in the art, and the purified product is sequenced, and then passed Observing whether the sequence obtained by sequencing has t...
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