Primer set and kit for detecting human ifitm5 gene mutation
A technology of primer sets and kits, which is applied in the field of primer sets and kits for detecting human IFITM5 gene mutations, and can solve problems such as inability to detect large fragment deletions, copy number variations, no clear candidate genes, and missing test results. Achieve cost and manpower savings, reduce testing costs, and ensure accuracy and specificity
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Embodiment 1
[0055] The DNA sequence of IFITM5 gene (NG_032892.1) was analyzed according to the NCBI GeneBank database, and the primers for exon amplification from 5'UTR to 3'UTR were designed. The amplified region includes all exons of IFITM5 and the intron sequence at the junction of exons and introns not less than 50 bp. The nucleotide sequences of primers used in PCR amplification are shown in Table 1.
[0056] Table 1 IFITM5 gene exon primer amplification and sequencing sequence
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Embodiment 2
[0060] A kit for detecting human IFITM5 gene mutations including 5'UTR and 3'UTR, comprising:
[0061] A primer set composed of the above PCR primers for detecting IFITM5 gene mutations including 5'UTR and 3'UTR;
[0062] PCR amplification reagents include: 10mM dNTP Mix, containing Mg 2+ 2×Phanta Max Buffer, Phanta MaxSuper-Fidelity DNA Polymerase, de-RNase and DNase water (DEPC water);
[0063] The working concentration of each component in the PCR amplification system is: 10000μM dNTP Mix, 2mM2×Phanta MaxBuffer, 1U / μl Phanta Max Super-Fidelity DNA Polymerase, both upstream and downstream primers are 5μM, and the template concentration is 50-400ng / μl.
[0064] The PCR product purification reagents include: 1U / μl SAP enzyme, 10U / μl ExoI enzyme, DEPC water; the working concentration of each component in the PCR product purification system is: 0.05U / μl SAP enzyme, 0.5U / μl ExoI enzyme.
[0065] The DNA sequencing reagents include: the above-mentioned sequencing primer set fo...
Embodiment 3
[0069] Utilize above-mentioned kit for detecting the IFITM5 gene mutation including 5'UTR and 3'UTR to carry out the human IFITM5 gene mutation detection method, the steps are as follows:
[0070] (1) Extraction of genomic DNA from peripheral blood
[0071] Collect 5ml of peripheral blood from the patient, use Omega Company DNA Extraction Kit (D3392-01) to extract whole blood genomic DNA, and use NanoDrop2000 / 2000c spectrophotometer to measure DNA concentration and purity; see the product manual for specific operating steps and conditions;
[0072] (2) PCR amplification
[0073] The PCR reaction system is 40 μl, including:
[0074] 10mM dNTP Mix 1μl, 2×Phanta Max Buffer 20μl, Phanta Max Super-FidelityDNA Polymerase 1μl, upstream primer 4μl, downstream primer 4μl, DNA template 50-400ng, make up to 40μl with DEPC water.
[0075] The PIC-200 type PCR instrument of BIO-RAD Company was used for landing PCR, and the PCR reaction conditions were as follows:
[0076] Pre-denaturati...
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