COL4A5 gene mutant and its application
A mutant and nucleic acid technology, applied in the field of COL4A5 gene mutants, can solve problems that need to be deepened
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Embodiment 1
[0286] Example 1 Determining AS Syndrome Causative Mutations
[0287] 1. Sample collection
[0288] The inventor collected a Chinese AS syndrome pedigree, the pedigree of which is shown in figure 2 . Such as figure 2 As shown, among them, means a normal male, represents a male patient, Denotes a female patient, the arrow points to the proband, and the proband is an identical twin.
[0289] The inventors collected peripheral blood samples from the above-mentioned AS syndrome patient family and normal persons in the family.
[0290] 2. Target region capture sequencing
[0291] The inventor used NimbleGen Sequence Capture Arrays combined with Illumina Hiseq2000 high-throughput sequencing technology to detect the COL4A3 (MIM: 120070 ), COL4A4 (MIM: 120131) and COL4A5 (MIM: 303630) gene coding region sequences were sequenced.
[0292] details as follows:
[0293] 2.1 DNA extraction
[0294] collection figure 2 From the peripheral blood of two probands (III-1 and I...
Embodiment 2
[0303] Example 2 Sanger method sequencing verification
[0304] The COL4A5 gene of the family members (I2, II1, II2, II4, III1, III2, III3) in the AS syndrome patient family described in Example 1 was detected: primers were designed for the c.1365_1373delTCCAGGCCC mutation of the COL4A5 gene, Then, the relevant sequence of the mutation site was obtained by PCR amplification, product purification and sequencing, and the COL4A5 gene and the correlation between the mutation and AS syndrome were verified according to whether the sequence was determined to be mutant or wild type.
[0305] The specific method steps are as follows:
[0306] 1. DNA extraction
[0307] 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.
[0308] 2. Primer design and PCR reaction
[0309] First, with reference to the human genome sequence database GRCh37 / hg19, the COL4A...
Embodiment 3
[0324] According to the above target region capture sequencing method, a sporadic patient outside the family was verified by target region capture sequencing, and no suspected pathogenic mutation was found in the entire coding region of the COL4A5 gene.
[0325] Then, the c.1365_1373delTCCAGGCCC mutation of the COL4A5 gene was detected in the database of 100 cases of unrelated normal controls of the same race, and the mutation was not detected, which proved that the mutation site was a very low-frequency mutation.
[0326] Thus, it is further proved that the c.1365_1373delTCCAGGCCC (p.456_458delPGP) mutation of the COL4A5 gene detected in Example 1 is the pathogenic mutation of AS syndrome.
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