Method for detecting and relatively quantifying aristolochic acid I-DNA adducts in tissue DNA
A technique for relative quantification of aristolochic acid, applied in the field of detection and relative quantification of aristolochic acid I-DNA adducts in tissue DNA, can solve problems such as inaccuracy, and achieve efficient, simple, accurate detection and relative quantification Effect
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Embodiment 1
[0030] Embodiment 1, preparation and identification of aristolochic acid I DNA adduct (dA-AL-I) standard substance
[0031] 1. Preparation of aristolochic acid I DNA adduct (dA-AL-I) standard
[0032] 1.1 Dissolve 2mg of dA in 1ml of 50mM potassium phosphate solution (pH 5.8), add 1mg of aristolochic acid I and mix well, add zinc powder, and react in the dark at 37°C for 16h.
[0033] 1.2 Centrifuge at 12000×g for 10 minutes, and take the supernatant.
[0034] 2. Identification of synthetic aristolochic acid I DNA adduct (dA-AL-I) standard by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)
[0035] 2.1 The synthetic dA-AL-I standard was identified by using ACQUITY UPLC system (Waters) coupled with XEVO-G2XS quadrupole time-of-flight mass spectrometer (UPLC-QTOF-MS, Waters). 7 μl of the standard was injected into an ACQUITY HSS T3 column (2.1 mm×100 mm, 1.8 μm, Waters), and the mobile phases A and B were 0.1% formic ac...
Embodiment 2
[0037] Embodiment 2, DNA is digested into single deoxynucleoside
[0038] 1. Extraction of Tissue DNA
[0039] Mince the liver tissue (or other tissues) of mice treated with aristolochic acid I or PBS and add to the solution containing 100mM EDTA, 10mM NaCl, 0.1% SDS, proteinase K (0.2mg / ml) and RNase A (0.2mg / ml ) of 10 mM Tris-HCl (pH 8.0). DNA was extracted by phenol / chloroform with 10mM MgCl 2 Dissolve in 5mM bis-Tris-HCl (pH 7.1).
[0040] 2. DNA digestion
[0041]2.1 The DNA is 1mg DNA / 1ml containing 10mM MgCl 2 5mM bis-Tris-HCl (pH 7.1) for preparation. First add deoxyribonuclease I (dissolved in 0.15M NaCl, 300U / mg DNA), mix well, incubate at 37°C for 1.5h, then add nuclease P 1 (dissolved in 1mM ZnCl 2 , 4U / mg DNA), mix well, incubate at 37°C for 3h, and finally add alkaline phosphatase (dissolved in 1mM MgCl 2 , 2U / mg DNA) and phosphodiesterase I (dissolved in 110mM NaCl, 15mM MgCl 2 and 110 1 mM Tris-HCl (pH 8.0), 0.0714 U / mg DNA) of 50% glycerol, and inc...
Embodiment 3
[0044] Detection and relative quantification of dA-AL-I in embodiment 3, DNA
[0045] 1 Detection and relative quantification of dA-AL-I in DNA hydrolysis products by ultra-high performance liquid chromatography / triple quadrupole mass spectrometry (UHPLC / QQQ MS, Waters)
[0046] 1.1 The chromatographic column, mobile phase and gradient elution program used in this system are the same as those used in the UPLC-QTOF-MS system. 2μl of standard and DNA digestion products were injected into the chromatographic column in sequence. The system also uses positive ion mode, triple quadrupole mass spectrometer conditions: capillary voltage, 0.5kV; cone voltage, 40V; ion source temperature, 150°C; desolvation temperature, 500°C; cone gas flow rate, 150l / h; desolvation gas flow rate, 1000 l / h; detection method: multiple reaction detection mode (MRM). The MRM ion pairs are 543.16 / 427.12, 543.16 / 395 and 543.16 / 292, and the corresponding collision energies are 25, 30 and 35eV, respective...
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