Reagent used for detecting forkhead transcription factor O1 (FOXO1) gene mutation and application
A technology of transcription factors and reagents, which is applied in the field of reagents for the detection of forkhead transcription factor O1 gene mutations, can solve the problems of affecting Wnt signaling pathway conduction and reduction, and achieve the effects of improving detection specificity, simple experimental operation, and good sensitivity
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Embodiment 1
[0038] [Example 1] Primer probe design
[0039] According to the FOXO1 mRNA sequence (NCBIReferenceSequence: NM_002015.3) reported by the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov), use the PrimerExpressSoftwareforReal-TimePCR software developed by AppliedBiosystems to design specific Primers and probes for FOXO1.
[0040] FOXO1R609C:
[0041] FOXO1-F:5'-CCTTTCTCCACCAGGAGAAGCTC-3'; (SEQ NO.1)
[0042] FOXO1-R:5'-CTTGACACTGTGTGGGAAGCTT-3'; (SEQ NO.2)
[0043] Mutant FOXO1(PNA)-P: 5'FAM-TGTTCATTGAGTGCTTAGAC-BHQ3'; (SEQNO.5)
[0044] Wild-type FOXO1-PNA: 5'-TGTTCATTGAGCGCTTAG-3'; (SEQNO.7)
[0045] Mutant target sequence:
[0046] 9()
[0047] Wild-type target sequence:
[0048] (SEQ NO. 10)
[0049] FOXO1D624N:
[0050] FOXO1-F:5'-CCTTTCTCCACCAGGAGAAGCTC-3'; (SEQ NO.3)
[0051] FOXO1-R:5'-CTTGACACTGTGTGGGAAGCTT-3'; (SEQ NO.4)
[0052] FOXO1(PNA)-P:5'FAM-AATGACCTCATGAATGGAGATA-BHQ3'; (SEQ NO.6)
[0053] FOXO1-PNA: AATGACCTCATGGA...
Embodiment 2
[0062] [Example 2] Construction of recombinant plasmids containing FOXO1 gene mutant and wild-type DNA fragments 1. Human endometrial cancer Ishikawa cell line culture and passage
[0063] 1) Cell culture
[0064] All cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C and 5% CO2.
[0065] 2) Cell passage
[0066] First, use a sterilized pipette to suck out the culture medium in the cell culture dish, add PBS buffer to wash twice, slowly add an appropriate amount of trypsin to the cells, wait until the cells become round, adjust the angle and the cells can move, then add 3ml of 10 DMEM medium with % fetal bovine serum, after repeatedly blowing gently, observe the cell morphology and count under the microscope, pass appropriate amount of cells to other sterilized culture dishes according to the content of cells in the culture dish, add 5ml of DMEM medium Then put into 5% CO2 incubator.
[0067]...
Embodiment 3
[0090] [Example 3] Amplified sample by real-time fluorescent quantitative PCR method
[0091] Take 1-5 μg of extracted total RNA and add it to PCR reaction solution, which includes: sterile water, DNA polymerase with 5'→3' exolytic activity, dNTPs, 10×PCRBuffer, RNASIN, M-MLV reverse transcriptase , oligo(dT) and a solution containing Mg ions. Among them, 0.3 μL of DNA polymerase with 5’→3’ exolytic activity at a concentration of 5 U / μL, 2 μL of dNTPs at a concentration of 10 mmol / L, 5 μL of 10×PCRBuffer, 0.6 μL of RNASIN at a concentration of 40 U / μL, and a concentration of 200 U / L μL of M-MLV reverse transcriptase 0.6 μL, concentration of 25mmol / L MgCl 2 Solution 5 μL, add sterile water to a volume of 50 μL. Wherein, the DNA polymerase with 5'→3' excision activity can be Taq enzyme.
[0092] PCR amplification: put each reaction tube into the reaction tank of the fluorescent quantitative PCR instrument, set the type of labeled fluorescent group, sample name and type, and s...
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