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  Section: Biotechnology Methods » Cell Biology and Genetics
 
 
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Karyotyping

 
     
 
Content
Cell Biology and Genetics
  Cell Cycles
  Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain
  Meiosis in Grasshopper Testis (Poecilocerus Pictus)
  Mitosis in Onion Root Tip (Allium Cepa)
  Differential Staining of Blood
  Buccal Epithelial Smear and Barr Body
  Vital Staining of DNA and RNA in Paramecium
  Induction of Polyploidy
  Mounting of Genitalia in Drosophila Melanogaster
  Mounting of Genitalia in the Silk Moth Bombyx Mori
  Mounting of the Sex Comb in Drosophila Melanogaster
  Mounting of the Mouth Parts of the Mosquito
  Normal Human Karyotyping
  Karyotyping
  Black and White Film Development and Printing for Karyotype Analysis
  Study of Drumsticks in the Neutrophils of Females
  Study of the Malaria Parasite
  Vital Staining of DNA and RNA in Paramecium
  Sex-Linked Inheritance in Drosophila Melanogaster
  Preparation of Somatic Chromosomes from Rat Bone Marrow
  Chromosomal Aberrations
  Study of Phenocopy
  Study of Mendelian Traits
  Estimation of Number of Erythrocytes [RBC] in Human Blood
  Estimation of Number of Leucocytes (WBC) in Human Blood
  Culturing Techniques and Handling of Flies
  Life Cycle of the Mosquito (Culex Pipiens)
  Life Cycle of the Silkworm (Bombyx Mori)
  Vital Staining of Earthworm Ovary
  Culturing and Observation of Paramecium
  Culturing and Staining of E.coli (Gram’s Staining)
  Breeding Experiments in Drosophila Melanogaster
  Preparation of Salivary Gland Chromosomes
  Observation of Mutants in Drosophila Melanogaster
  ABO Blood Grouping and Rh Factor in Humans
  Determination of Blood Group and Rh Factor
  Demonstration of the Law of Independent Assortment
  Demonstration of Law of Segregation

Principle
Karyotyping is a valuable research tool used to determine the chromosome complement within cultured cells. It is important to keep in mind that karyotypes evolve with continued culture. Because of this evolution, it is important for the interpretation of biochemical or other data, that the karyotype of a specific subline be determined. Numerous different technical procedures have been reported that produce banding patterns on metaphase chromosomes. A band is defined as the part of a chromosome that is clearly distinguishable from its adjacent segments by appearing darker or lighter. The chromosomes are visualized as consisting of a continuous series of light and dark bands. A Gstaining method resulting in G-bands uses a Giemsa or Leishman dye mixture as the staining agent. What follows is a brief description of the steps involved in assembling a karyotype.

Time Required
15–30 minutes to cut, arrange, glue and interpret 1 metaphase spread on a karyotype sheet.

Procedure
  1. Count the number of chromosomes. Solid-stained chromosomes or chromosomes treated with a trypsin and giemsa stain can be counted at the microscope with a 100X magnification. However, for analysis such as identification of marker chromosomes or determination of the number of copies of individual chromosomes, it is usually necessary to photograph and print the chromosome spreads. Refer to procedures in this manual for
    black and white photography and film development. Two prints should be made of each spread. One will be cut for karyotyping; the uncut print serves as a reference if questions arise about the interpretation of a certain
    chromosome.
  2. Cut out each individual chromosome and arrange on a karyotype sheet. Chromosomes are ordered by their length, the position of the centromere, the position of the chromosome bands, and the relative band sizes and
    distributions.
  3. In the construction of the karyotype, the autosomes are numbered 1 to 22, in descending order of length. The sex chromosomes are referred to as X and Y. The symbols p and q are used to designate, respectively, the short and long arms of each chromosome. There are 7 groups identified in the karyotype, and data pertaining to each group.
  4. Secure chromosomes in place with glue. Pair the chromosomes closely together and align the centromeres (for easier band comparison and checking for structural chromosome aberrations). If possible, have a second
    technologist check the interpretation of the karyotype before chromosomes are secured in place.
  5. A description of the karyotype should be recorded on the karyotype sheet. First record the number of chromosomes, including the sex chromosomes, followed by a comma (,). The sex chromosome constitution is given next. Any structural rearrangements and additional or missing chromosomes are listed next. Other information such as the cell line number, the date karyotype was prepared, the specimen type, and the technologist should also be recorded on the karyotype sheet.

 
     
 
 
     




     
 
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