Primer for detecting CREBBP (cAMP-Response Element Binding Protein) gene mutation site, kit and application
A kit and site technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as no effective detection, and achieve good application prospects and great clinical application value
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Embodiment 1
[0028] Design of primers for detection of CREBBP gene mutation sites
[0029] Determine the gene sequence of the CREBBP gene CDS region (4450T>G) site on NCBI, and use Primer Premier5.0 software to design amplification primers for the DNA fragment including this site (upstream primer 5'-CTTAAAGGCAGGGCCGATTT-3'; Downstream primers: 5'-TATGCGAATGCAAGAA AAAGGCA-3'), to ensure that the length of the fragments of the amplified products after enzyme digestion differs by more than 100bp, so that the agarose gel electrophoresis can be distinguished.
[0030] Specifically, the primers are a pair of amplification primers, including an upstream primer F1 and a downstream primer R1, wherein the base sequence of the upstream primer F1 is shown in the sequence listing SEQ ID NO: 1, and the base sequence of the downstream primer R1 is shown in the sequence listing Shown in SEQ ID NO:2.
[0031] The primers are used for restriction endonuclease fragment length polymorphism polymerase chain r...
Embodiment 2
[0033] Kit design for detection of CREBBP gene mutation sites
[0034] The kit for detecting the CREBBP gene mutation site provided in this example includes the primers in Example 1.
[0035] Further, the kit for detecting the CREBBP gene mutation site provided in this example includes the primers and restriction endonucleases in Example 1.
[0036] Primer Premier 5.0 software was used to analyze the recognition of endonucleases, and it was determined that restriction endonucleases could specifically recognize this site.
[0037] In this example, the restriction endonuclease is MboII.
Embodiment 3
[0039] The primers in Example 1 and the kit in Example 2 were used to detect the 4450th T>G site mutation in the CDS region of the CREBBP gene and to predict the correlation between relapsed follicular lymphoma and the curative effect of chidamide Research
[0040] (1) Use restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) to detect the CREBBP gene CDS region (4450T>G) site;
[0041] (2) Collect tumor tissue samples from 2 patients with relapsed FL (Table 1), divide the patients into groups that respond to chidamide and those that do not. After grinding the tissues, DNA is extracted using QIAGEN kits;
[0042] (3) Using the primers in Example 1 and the kit in Example 2, amplify the tumor tissue DNA of 2 cases of recurrent FL patients as a template to obtain PCR products, and determine that the enzyme digestion reaction system is 30 μL, including the following components: PCR product 14μL; 10 times buffer solution (NEB company Buffer) 3 μL; MboII r...
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