DNA Replication and BrdU Staining in Mitotic Chromosomes
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DNA replication, BrdU staining, mitotic chromosomes, cell cycle, S phase, G1 phase, G2 phase, interphase, mitosis
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Abstract
This document explains the staining of mitotic chromosomes using bromodeoxyuridine (BrdU) and Giemsa dye, indicating the precise time and cell cycle stage of observation. It also describes the process of DNA replication and the incorporation of BrdU into DNA strands.
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[...] This description corresponds to the observation on the photograph which was therefore taken in metaphase of 26th mitosis Represent on a scheme, the staining obtained on the chromosomes if an additional cycle is performed on a culture medium devoid of thymine but containing BrdU: Metaphase 1 mitosis ?TATGGATAGCTTTAAA? ?ABrACCBrABrCGAAABrBrBr? ?ABrACCBrABrCGAAABrBrBr? ?TATGGATAGCTTTAAA Metaphase 2ème mitosis ?BrABrGGABrAGCBrBrBrAAA? ?ABrACCBrABrCGAAABrBrBr? ?ABrACCBrABrCGAAABrBrBr? ?TATGGATAGCTTTAAA Metaphase 3ème mitosis ?BrABrGGABrAGCBrBrBrAAA? ?ABrACCBrABrCGAAABrBrBr? ?ABrACCBrABrCGAAABrBrBr? ?BrABrGGABrAGCBrBrBrAAA? The chromatids will all have 2 strands marked by BrDU and all chromosomes will be colored in gray. [...]
[...] CTTTAAAT In G2, the DNA is entirely duplicated and 2 identical molecules form the metaphasic chromosomes: ?TATGGATAGCTTTAAA ?ABrACCBrABrCGAAABrBrBr? ?ABrACCBrABrCGAAABrBrBr? ?TATGGATAGCTTTAAA We have 2 black chromatids so far. However, we need both strands to have incorporated BrdU for the chromatid to be gray. At the end of 1 mitosis, the chromatids separate and each of the 2 daughter cells presents 1 black chromosome and 1 gray chromosome. If the culture on BrdU-containing medium is prolonged, the two new strands will also be marked and we will therefore have 2 different metaphase chromosomes: one will have 2 BrdU strands and will be gray and the other will have only one and will be non-colored. [...]
[...] The cell cycle is therefore the set of steps of a cell's life with phases: interphase (divided into G1, G2) and mitosis. DNA replication occurs during the S phase. This DNA duplication or replication occurs by opening the double DNA chain. In fact, the adenine-thymine and guanine-cytosine bases of the two strands are separated and new nucleotides are incorporated onto each strand by complementarity. The two newly formed molecules thus constitute the 2 chromatids of the chromosome in metaphase. Before the culture in a medium containing BrdU, in interphase, the DNA molecule presented itself as follows: ?TATGGATAGCAAATTT? [...]
