Non-Programmed DNA Synthesis Detection Experiment
Non-Programmed DNA Synthesis Detection Experiment
Unscheduled DNA Synthesis (UDS) is DNA synthesis outside of S phase. In general, intracellular DNA synthesis is only seen in S phase, but when the cellular DNA is damaged in non-S phase, DNA synthesis also occurs as the DNA damage is repaired, i.e., UDS.
Operation method
Non-programmed DNA synthesis detection experiments
Principle
Unscheduled DNA Synthesis (UDS) is DNA synthesis outside of S phase. Under normal circumstances, intracellular DNA synthesis is only seen in S phase, but when the DNA of cells in non-S phase is damaged, DNA synthesis phenomenon, i.e., UDS, also occurs with the repair of DNA damage, and UDS is also induced in the DNA when cellular DNA damage is induced by the action of chemical mutagens and carcinogens. Therefore, UDS is one of the short-term test methods for detecting environmental mutagens and carcinogens. When isotope-labeled DNA precursors are added to the culture environment, the damaged DNA can be detected by isotope technology because the DNA precursors can be incorporated into the DNA strand in a semiconservative manner outside the DNA synthesis period, even if the test substance is mutagenic or carcinogenic.
Materials and Instruments
Cells Move Human diploid fibroblasts W1 38 as an example Caveat I. Factors affecting UDS1. BackgroundUnlike S-phase DNA synthesis, the amount of UDS is very low, and the background must be controlled at a comparable level to show UDS.The key to controlling background is to reduce S-phase DNA synthesis; incubation with arginine-deficient Eagle's solution and treatment with hydroxyurea can accomplishThis can be achieved by incubation with arginine-deficient Eagle's solution and hydroxyurea treatment.Even after these treatments, there is still a certain amount of background, and the sources include(1) Semi-conserved replication in a few S-phase cells;(2) Mild microbial contamination； （3） DNA synthesis in cytoplasmic mitochondria； （4） natural repair； （5） 3H-TdR nonspecific attachment； （6） radiochemical impurities； （7） reuse of thymine catalysts.To control background, in addition to reducing or eliminating the effects of the above factors, it is worth noting that hydroxyurea, when used in excessive amountswill also inhibit UDS and have the effect of interfering with the substance to be examined. Therefore, the concentration and duration of hydroxyurea should be selected according to the different cellTherefore, the concentration and duration of hydroxyurea should be chosen according to different cells, so that the effect of known positive carcinogens can be shown at least when hydroxyurea inhibits the UDS. 2. Drug propertiesDifferent mutagens act on cells in different ways and do not induce the same type of DNA damage. Therefore, the dosage and duration of action of different drugsTherefore, the dosage and duration of action of different drugs are also different.For example, nitrogen mustard at 10-4M induces significant repair synthesis in lymphocytes within 2 hours, while MNM at 10-4M induces significant repair synthesis in lymphocytes within 2 hours.For example, nitrogen mustard at 10-4 M induces significant repair synthesis in lymphocytes within 2 hours, whereas MNNG induces UDS at a peak of 12 hours after its action.Detection of different drugs, especiallyWhen testing different drugs, especially environmental carcinogens, a dose-response curve should be made to select the optimal time to show UDS, and metabolic activation should also be taken into account to avoid false-negative results.The metabolic activation effect should also be taken into account to avoid false-negative results. 3. CellsWhen testing the DNA repair ability of experimental cells. The results are expressed as the average number of silver particles/nucleus or as the number of nuclei containing about 10 silver particles.The results are expressed as the mean number of silver grains/nucleus or as the percentage of cells containing about 10 silver grains. When performing experiments with diploid cells, karyotype analysis should be performed first. Common Problems I. Suitable cells are required for UDS experiments. Ideal cells that can express the UDS instructions should have 1. the ability to repair DNA damage; For more product details, please visit Aladdin Scientific website.
EMEM Hydroxyurea Trypsin MNNG
I. Experimental combinations
In order to confirm the exact role of the test substance, the following experimental combination should be made
Determination of UDS by autoradiography
1. Cell culture
WI-38 fibroblasts, complete culture medium support cover slip culture method, cell growth to Confluence;
2. Inhibition of DNA synthesis
Discard the old culture medium, add arginine-free EMEM culture medium, and continue to incubate for 20 to 24 hours. Discard old culture medium, add arginine-free EMEM culture medium and continue incubation for 20 to 24 hours;
3. add test substance
Add 10 mM hydroxyurea (plus MNNG for positive control) and incubate for 2 hours;
4. H-TdR treatment
2, 3, 4 Add 3H-TdR (5 μC/per dish) in each dish and incubate for 20-24 hours;
5. Rinsing
Remove the culture medium and rinse with calcium- and magnesium-free BSS;
6. Preparation
Preparation of radioautographic specimen method (refer to in situ hybridization gene localization method) Fixed cells, coated with glue, exposure, development and staining;
7. Observation
Counting under the oil microscope, first of all, exclude the S-phase semiconserved replicating cells (for the cells densely covered by silver particles Nuclei), only the number of silver particles on the nucleus is counted, and then subtract the background (30-50 cells in each case), the result will be calculated as The result is expressed as the mean value of the number of silver grains/nucleus or the percentage of cells containing about 10 silver grains.
Liquid flash counting method
1. Cell culture and treatment
Human diploid fibroblasts: inoculate in a petri dish. 2;
2. add culture medium, MNNG, hydroxyurea, and 3H-TdR treatment, etc. The same as the previous 5 items, since 5 rinsed;
3. digestion
Digested with 0.25 % trypsin and made into suspension;
4. Liquid flash counting specimen preparation
10 % trichloroacetic acid treatment, collect the cells on a glass fiber filter paper, anhydrous The cells were collected on glass fiber filters, dehydrated and dried with ethanol, placed in a liquid flash cup, added scintillation solution and counted on a liquid flash counter; the results were reported as dpm/106 cells or cpm/cell per dish. The results were expressed as dpm/106 cells or cpm/cell per dish.
2. the ability to metabolize activated carcinogens;
3. a large proportion of interphase cells and easy access to materials. Properties.
However, not every cell has the above requirements.
In fact, each cell has its own advantages and disadvantages. The following types of cells are commonly used The following types of cells are commonly used
1. Primary cultured rat hepatocytes
They have the ability to metabolize activated carcinogens and repair DNA damage, but the ratio of S-phase cells is large, and they are suitable for displaying UDS by radiographic autoradiography. However, the ratio of S-phase cells is large, which is suitable for displaying UDS by autoradiography, and should not be used for liquid flash counting.
2. human peripheral blood lymphocytes
The majority of cells in quiescent phase are favorable conditions for UDS, but there are differences between individuals when screening for carcinogens. However, there is a problem of inter-individual variation when screening for carcinogens.
3. human diploid fibroblasts
WI-38 (ATCCCCL N075) is available, which can proliferate rapidly and in large numbers, and after cell confluence and monolayer synthesis, contact inhibition and cell arrest occur. After cell confluence and monolayer synthesis, contact inhibition, cell growth cessation, and cessation of DNA synthesis occur; UDS results can be obtained satisfactorily by either autoradiography or liquid-flash counting. It is an ideal cell with satisfactory UDS results by autoradiography or liquid flash counting, but it cannot metabolize the activated carcinogens.