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Chromosome karyotype analysis has a wide range of applications in genetic research, particularly in the clinical diagnosis of human genome diseases, as well as in the detection of genetic and genetic variations in organisms. It is a basic method for studying the heredity and variation of organism, the evolution of chromosome and the relationship of species. Mastering the technique of chromosome karyotype analysis plays an important role in the correct interpretation of genetic diversity. Background KARYOTYPE: The phenotype of the MITOTIC metaphase genome of an organism that is the sum of its List of organisms by chromosome count, size, and morphological characteristics.
mitotic metaphase genome Somatic cells have two identical sets of chromosomes, called sex chromosomes, which are expressed as autosomes (2n) and are directly related to sex. Each configuration has a set of chromosomes called haploids, which are denoted by N, and a diploid cell is formed when the amphoteric gametes combine. Karyotype analysis: The process of pairing, grouping, classifying and numbering the chromosomes in the nucleus according to the chromosome number, size, centromere position, arm ratio, secondary constriction, satellite and other morphological characteristics. KARYOGRAM: The karyotype of a species in which all the chromosomes of the genome are arranged according to their length, shape and type. Chromosome morphological classification: The centromere on both sides of the part known as the chromosome arm. If the arms are equal, they are called the isoarm chromosomes. If the arms are not equal, they are called the long arm and the short arm. According to the different positions of Centromere, the chromosomes can be divided into three types: Middle Centromere (m) , sub-middle Centromere (SM) , sub-end Centromere (St) and end Centromere (t) . Some chromosomes have satellite and secondary constriction. The common morphological indexes used to distinguish the chromosomes of each pair are: measuring the length of chromosomes, determining the position of Centromere, vice-constriction and presence or absence of satellite. The length of chromosome is not fixed and is influenced by heredity and environment, but it is generally believed that the elongation and shortening of each chromosome in a cell are the same. Therefore, the relative length is generally used to represent the length of each chromosome. The metaphase of cell division is the most typical period of chromosome morphological structure. It is used for karyotype analysis by selecting the well-stretched, well-defined and representative cell division phase. Experimental principle Chromosome preparation is the key to the core analysis, which is usually done using peripheral blood lymphocyte. Normally, human peripheral blood lymphocyte do not divide, but Phytohemagglutinin (Pha) can stimulate the blood lymphocyte into lymphoblast cells and restore their proliferative ability. Therefore, peripheral venous blood can be taken for short term culture and cultured until 72 hours when the cells enter a period of vigorous proliferation, when the addition of Colchicine inhibits cell division and stops cell division in the intermediate stage to obtain sufficient number of mitotic cells, karyotype analysis was performed by low permeability, fixation, sectioning and staining. Experimental Reagent 1640 complete medium, phytohemagglutinin (Pha) , Colchicine (10 MG/ML) , KCL hypotonic solution (0.075 mol/l) , methanol, glacial acetic acid, Giemsa solution and phosphoric acid buffer solution (PH6.8) . Experimental Procedure 1. The peripheral blood was cultured in an incubator at 37 °C for 72 hours. 2. At the end of 68H, colchicine concentration reached 0.025 μg/ml in the culture flask, added Colchicine, and the cell division stopped in the middle phase in the incubator. 3. PREPARATION OF CHROMOSOME SLIDES: 3.1 cells were collected: After homogenizing the culture with a straw, they were transferred into a 5 ml centrifuge tube, centrifuged for 1000 R/min, centrifuged for 10 Min, supernatant removed, and cell precipitate remained. 3.2 low-permeability treatment: adding 0.075 M KCL LOW-PERMEABILITY SOLUTION ABOUT 5 ml at 37 °C pre-temperature. The cells were suspended in the hypotonic solution and treated in 37 °c water bath for 20 Min to disintegrate the red blood cells. 3.3 pre-fixation; add the newly formulated methanol: ACETIC ACID (3∶1) fixative solution about 1ml, gently beat well. 3.4 centrifugation: 1000r/min, Centrifugation for 10Min, supernatant removed. 3.5 first fixation: adding about 5 ml of fixative liquid along the pipe wall, gently beating with Straw, leaving at room temperature for 15 min, centrifuging for 1000 R/MIN, removing supernatant. 3.6 second fixation: 5 Ml of fixative liquid was added, then fixed for 15 min; 1000 R/min, centrifuged for 10 min, supernatant removed. 3.7 slice: add the fixative solution to 0.4 ml or so, carefully mix to form cell suspension. Two to three drops of cell suspension were added to each of the pre-frozen slides, and the slides were fixed by flame. 3.8 staining: GIEMSA solution (9∶1) was diluted with PH7.4 phosphoric acid buffer, and one prepared chromosome specimen was stained for 15ー20 Min, rinsed with water and air dried. 3.9 observation: The well-dispersed metaphase phase was selected at low power microscope, and then observed by oil microscope. 3.10 sealed slides: If the prepared slides need to be kept for a longer period of time, they can be sealed with sealant to prevent contact with the outside world. 4. Take pictures:
mitotic metaphase genome in the microscope The prepared specimen was placed under the objective lens and the microscope was adjusted well. The metaphase phase of cell division was found and the well-dispersed metaphase phase was selected. 5. LIST OF ORGANISMS BY CHROMOSOME COUNT: A number of well-dispersed dividing cells (30 ~ 50) can be counted. 6. Recording and measurement of morphological characteristics Chromosome karyotype analysis system: At present, there are many kinds of chromosome karyotype analysis systems, which can be used in chromosome karyotype analysis. The karyotype analysis system can automatically detect and classify chromosomes for research or diagnosis. Common Analysis Systems include: Videotest chromosome karyotype analysis software, MetaScan Karyotyping System FINDERTM KARYOTM module, IMSTAR automatic intelligent chromosome karyotype analysis System Pathkaryo series, etc. . Time to knock on the blackboard (1) karyotype analysis is not only suitable for peripheral blood cells, but also for normal cells. The key is to control the cell division time and determine the cell phase. (2) the fixation procedure is very important. Only one fixation can be omitted. The chromosome picture will be more complete if the chromosome is fixed twice. (3) the degree of inclination of the slides and the degree of wet cooling of the slides will directly affect the degree of chromosome dispersion.
Karyotyping analysis result |
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