Sister chromatid exchange specimen preparation and analysis experiments
Sister chromatid exchange specimen preparation and analysis experiments
Sister chromatids are two parallel chromatids linked by a single mitotic site, formed by replication of the same chromosome during interphase of cell division, existing in pairs during interphase, prophase, and midphase of cell division, and are identical in size, shape, structure, and origin.
Operation method
basic program
Principle
5-Bromodeoxyuridine nucleoside (5-Bromodeoxyuridinc or BrdU) is an analog of thymine nucleoside. When human lymphocytes undergo DNA replication in BrdU-containing cultures, BrdU exclusively replaces thymine nucleosides and is incorporated into the newly replicated DNA nucleotide strand. Thus, in just two replication cycles, one strand of the DNA of one of the sister chromatids can be BrdU-doped, and both strands of the double-stranded DNA of the other chromatid can be BrdU-doped. because of the change in the conformation of the DNA molecule in which both strands of the chromatid contain BrdU, this chromatid has a lower affinity for certain stains, and can be clearly seen in the staining of the chromatid with Giemsa stains. When stained with Giemsa stain, it is clear that the monomer composed of the DNA strand containing BrdU in both strands is lightly colored, while the other monomer is deeply colored. This technique can be used to check for two sister chromatid exchanges (SCEs) on the same chromosome. It has been shown that many mutagenic and carcinogenic agents can induce sister chromatid interchanges and induce chromosome breaks and rearrangements. SCE is much more sensitive to these factors than chromosomal aberrations. Therefore, the detection of sister chromatid exchange frequency is a new method to check the cell biological effects of carcinogenic and mutagenic agents, which has the advantages of sensitivity, accuracy, simplicity and rapidity.
Materials and Instruments
Human peripheral blood Move 1. Preparation of culture fluid, blood collection, culture and other operations are the same as those for the preparation of routine chromosomes. (See Methods of Preparation of Routine Chromosome Specimens.) For more product details, please visit Aladdin Scientific website.
BrdU solution SSC Giemsa stain PBS
UV lamp Constant temperature water bath incubator Petri dish Mirror paper
2. Add 0.2 ml of BrdU solution to the peripheral blood culture for 24 hours, so that the final concentration is 8 micrograms per milliliter of culture solution. After adding BrdU, wrap the culture bottle with black paper to praise the culture bottle, and put it in a 37℃ warm box to continue to cultivate for 48 hours.
3. Colchicine treatment is the same as before (see Preparation of chromosome specimens).
4. Collect cells, fixation, hypotonicity, preparation of slices in the usual way, and bake the specimen slices in a 37℃ warm box for 24 hours.
5. Place the specimen face up flat in the staining bath and add 2xSSC solution to the extent that the solution does not exceed the surface of the specimen. The specimen is then covered with a piece of microscope paper slightly larger than the specimen so that the edge of the paper hangs down into the 2xSSC solution to keep the specimen moist.
6. the staining bath in a 55 ℃ constant temperature incubator, on the 30 W ultraviolet lamp vertically illuminated specimens for 30 minutes, the lamp distance from the specimen is about 10 cm. irradiation, gently remove the mirror paper, and immediately rinse the specimen with distilled water. (The water temperature was about 40°C).
7. Stain the specimen with Giemsa staining solution (the original solution was prepared with pH 6.8 phosphate buffer 1:10) for about 5~10 minutes, rinse with tap water, air-dry and then microscopically examine the specimen.
8. Microscopic analysis of sister chromatid interchange and frequency calculation
(1) Under normal or abnormal conditions, the frequency of sister chromatid exchange is different. According to the obvious color difference of chromosomes in the above specimens, the frequency of sister chromatid exchange can be accurately detected by counting.
(2) Select chromosomes with good chromosome dispersion and two sister chromatids stained in a dark and light mid-phase. Where sister chromatid interchange occurs, light stained fragments can be seen on the dark stained chromatids. Interchange at chromosome ends was counted once (one SCE); interchanges in the middle of the chromosome were counted twice (two SCEs); and interchanges at the site of the mitre, which were judged not to be the result of a reversal of the two chromosomes, were counted once (one SCE).
(3) The frequency of SCEs was calculated after counting 30 SCEs in the mid-division phase.
(1) SCE frequency = sum of n midphase SCEs/n cells
② Normal SCE frequency in Chinese is 5.7 ± 0.4