Sister chromatid exchange (SCE) is a special type of homologous recombination between two chromatids formed during the replication of the same chromosome.The mechanism of SCE is not clear, but the frequency of SCE can reflect the degree of cellular damage during DNA synthesis.
Principle
The basic principle of the sister chromatid exchange assay is that 5-bromodeoxyuridine (5-BrdU) is an analog of deoxythymidine.
When human peripheral blood lymphocytes proliferate in a culture medium containing 5-BrdU, 5-BrdU replaces thymine in the newly replicated DNA strand. After two replication cycles, the DNA double strands of the two monomers of their intermediate chromosomes differed in chemical composition, i.e., BrdU was incorporated into both strands of one DNA double strand, whereas BrdU was incorporated into only one strand of the other DNA double strand.
By using special differentiation staining techniques on chromosome specimens, chromosomes with BrdU doping on both strands are lightly stained, while those with BrdU doping on only one strand are darkly stained. Therefore, when homologous fragments are exchanged between sister chromatids, the exchanged fragments can be seen with clear boundaries and symmetrical color shades at the interchanges.
Operation method
sister chromatid exchange assay
Principle
The basic principle of the sister chromatid exchange assay is that 5-bromodeoxyuridine nucleotide (5-BrdU) is an analog of deoxythymine nucleoside. When human peripheral blood lymphocytes proliferate in a culture medium containing 5-BrdU, 5-BrdU replaces thymine in the newly replicated DN A subchain. After two replication cycles, the DNA double strands of the two monomers of their intermediate chromosomes differ in chemical composition, i.e., BrdU is incorporated into both strands of one DNA double strand, whereas only one strand of the other DNA double strand is incorporated with BrdU. By using special differentiation staining techniques on chromosome specimens, chromosomes with BrdU doping on both strands are lightly stained, while those with BrdU doping on only one strand are darkly stained. Therefore, when homologous fragments are exchanged between sister chromatids, the exchanged fragments can be seen with clear boundaries and symmetrical color shades at the interchanges.
Materials and Instruments
Equipment: Move The basic procedure of sister chromatid exchange assay can be divided into the following steps: A Peripheral venous blood was collected by the semi-micro-volume method, inoculated with 0.5 mL of GI-1640 culture medium (5 mL) containing PHA, and incubated at 37 ℃ in a constant temperature incubator. B After 24 hours of incubation, add 5-BrdU (final concentration 10 μg/mL) into the culture bottle and mix gently. C. Wrap the culture bottle with black paper and incubate at 37 ℃ for 48 h in a constant temperature incubator protected from light. D Add colchicine (final concentration: 0.2 μg/mL) before harvesting, and continue to incubate for 2 hours. E Conventional method of preparation (see basic plan 1, chromosome specimen preparation), the prepared chromosome slide specimens were placed at room temperature for 2-3 d. The day before differentiation staining, the chromosome slide specimens were placed at room temperature. F The day before differentiation staining, place the chromosome slide specimens at 37 ℃ overnight. G Place two toothpicks parallel to each other in a 9 cm dish, place the aged chromosome slide specimen on the toothpicks, and then add an appropriate amount of 2 x SSC solution (to the extent of not exceeding the specimen). H Cover the specimen with a piece of microscope paper slightly wider than the slide, so that the edge of the paper is immersed in the 2 x SSC solution and the 2 x SSC solution seeps onto the chromosome slide specimen, keeping the specimen moist. I Place the petri dish on a 55 °C water bath stand with only the bottom of the dish touching the water. J Using 20 W ultraviolet lamp vertical irradiation specimen 30 min, irradiation distance 10 cm. K After irradiation, gently remove the microscope paper with tweezers and gently rinse the chromosome slide specimen with distilled water. L Stain the specimen with 4% Giemsa's staining solution (prepared with pH 6.8 phosphate buffer 20 min before use) for 5~10 min. M Gently rinse the chromosome slide specimens with distilled water and air dry. Caveat 1 5-BrdU should be dispensed as needed and stored at 4 ℃ away from light.2 Split phases in different proliferative cycles can be seen in the field of view. Chromosomes in the first proliferative cycle have only one strand of 5-BrdU in the DNA double strand of each chromosome, and the two sister chromatids are darkly stained; whereas in the split phase of the third proliferative cycle, the DNA double strand of each chromosome is doped with 5-BrdU, and the two chromatids are lightly stained. The above two phases are not suitable for SCE observation and counting. You should choose the split phase in the second proliferation cycle, that is, the split phase in which the two chromosomes of each chromosome are differently colored.3 When determining the exchange of chromosomes at the mitosis, it is necessary to exclude the occurrence of chromosome distortion here. For more product details, please visit Aladdin Scientific website.
① Optical microscope
② Constant temperature incubator
③ Constant temperature water bath
④ Blood collection equipment
⑤ Alcohol lamp
⑥ Culture bottle
⑦ Nipple pipette
⑧ Slides
⑨ 20 W ultraviolet lamp
⑩ Staining tank
⑪ 9 cm Petri dish
⑫ Human peripheral venous blood.
Reagents:
① GI-1640 medium
② Phytohemagglutinin (PHA)
③ Colchicine (100 μg/mL)
③ Colchicine (100 μg/mL) ④ 5-BrdU (100 μg/mL)
⑤ Hypotonic solution (0.075 mol/L KCI solution)
⑥ 2 x SSC
⑦ Jimsa stock solution
⑧ Phosphate buffer (pH 6.8)
⑨ Methanol
⑩ Glacial acetic acid