GATA-2 mutator as one of leukemia rapid change major genes and its use
A GATA-2, mutation gene technology, applied in the fields of application, gene therapy, genetic engineering, etc., can solve the problems of few reports, no GATA-2 mutation and chronic myelogenous leukemia blast
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Embodiment 1
[0060] PCR amplification of each exon of GATA-2 gene
[0061] 1. Patient sample collection and processing: Collect 5-10ml of the patient's bone marrow before treatment begins, anticoagulate with heparin, separate mononuclear cells by density gradient centrifugation on the same day, add appropriate amount of white blood cell lysate and proteinase K, and wait for the white blood cells to be fully lysed Genomic DNA was extracted by the phenol-chloroform method and stored at -20°C for later use. Total RNA extraction and cDNA synthesis: Total RNA extraction and reverse transcription were carried out according to the standard operating procedures of the kit (GIBCO BRL Company).
[0062] Normal control: 5ml of peripheral blood from employees whose annual physical examination indicators were normal in Ruijin Hospital, a total of 200 copies. DNA was prepared as previously described.
[0063] 2. Primer design: The software Primer premier 5 was used, and the primer parameters were set ...
Embodiment 2
[0067] PCR product purification and sequencing:
[0068] 1. PCR product purification Use Shrimp Alkaline Phosphatase (SAP) and exonuclease (Exonuclease I) to purify the PCR product, the reaction system is: SAP 0.16 μl, Exonuclease I 0.25 μl, PCR product 50ng, add water to make up to the total volume 7μl; purification reaction program: 37°C for 60min, 80°C for 15min, 4°C forver.
[0069] 2. ABI 3700 Automatic Sequencer (PEBiosystems) was used for the sequencing of PCR purified products. Sequence the PCR fragments
[0070] 1) Take out the PCR purified product, MIX, and 0.8uM primers from the refrigerator at -20°C
[0071] 2) Take a 96-well reaction plate, mark the name of the template (that is, the name of the PCR purified product) and the date, add 2 ul of primers one by one with a continuous sampler, then add 2 ul of the template with a 12-hole row gun, and finally add mix one by one with a continuous sampler Shake 2ul on the centrifuge and stop when it reaches about 1300 r...
Embodiment 3
[0081] Search for mutation sites:
[0082] Polyphred software was used to search for mutation sites. The Polyphred software will mark suspicious mutation sites with various colors, and different colors represent different degrees of confidence. The final confirmation needs to be evaluated with the naked eye by opening the graphics. For mutation sites with high abundance, the reliability of software identification is very high, but for mutation sites with low abundance, if only one sample shows a heterozygous pattern, it needs to be determined with the naked eye based on the evaluation criteria to remove the Bad interference. To judge whether a site is a mutation site, at least the following conditions should be met: 1) It shows that the sequences on both sides of the heterozygous base are identifiable single peaks, and the background is relatively low; 3) Comparing the sequencing graphs of heterozygous individuals and homozygous individuals, the base peaks in the graphs of h...
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