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  1. 1. Karyotyping and Chromosomal banding
  2. 2. Study Objective <ul><li>Study objective 1 : To understand the structure of the chromosomes </li></ul><ul><li>Study objective 2: to understand the various banding techniques and their application </li></ul>
  3. 3. Karyotype <ul><li>The karyotype is the complete chromosomal set of the nucleus of the cell. A diagrammatic representation of the karyotype with all of the pairs of chromosomes arranged in order of size iscalled an ideogram. </li></ul><ul><li>Every organism has a standard karyotype, which provides a frame of reference for the analysis of mutations. </li></ul>
  4. 4. Karyotype (contd..) <ul><li>Karyotypic analysis is the study of all of the visible traits of chromosomes in a typical cell. This is useful in studying speciation. </li></ul><ul><li>Let’s consider an example: </li></ul><ul><li>Ex 1: distinct species of Drosophila that are very similar in appearance are distinguished easily by karyotypic analysis, which reveals numerous chromosomal inversions and translocations. </li></ul>
  5. 5. Chromosome structure <ul><li>Composed of chromatin, a combination of nuclear DNA and proteins </li></ul><ul><li>For karyotyping cells are captured in metaphase </li></ul><ul><li>metaphase stage in mitosis at which chromosomes are aligned along the cell equator </li></ul>
  6. 6. Chromosomal Banding <ul><li>A chromosome banding pattern is comprised of alternating light and dark stripes, or bands, that appear along its length after being stained with a dye. </li></ul><ul><li>A unique banding pattern is used to identify each chromosome and to diagnose chromosomal aberrations </li></ul><ul><li>chromosome breakage </li></ul><ul><li>loss </li></ul><ul><li>duplication or inverted segments. </li></ul>
  7. 7. Chromosomal Banding (contd..) <ul><li>In the 1950s, chromosomes from the cell’s nucleus were identified with a uniform (unbanded) stain that allowed for the observation of the overall length and primary constriction (centromere) of each chromosome, as well as a secondary constriction in chromosomes 1, 9, </li></ul><ul><li>16 and the acrocentrics (chromosomes whose centromeres are near the tips). </li></ul><ul><li>The staining techniques used to make the bands visible were developed in the late 1960s and early 1970s . </li></ul>
  8. 8. Q-Banding Technique <ul><li>Developed by T. Caspersson and his colleagues </li></ul><ul><li>Quinacrine mustard, an alkylating agent, was the first chemical to be used for chromosome banding </li></ul><ul><li>They observed alternating bright and dull bands under fluorocent microscope </li></ul><ul><li>Quinacrine-bright bands were composed primarily of DNA that was rich in the bases adenine and thymine </li></ul><ul><li>Quinacrine-dull bands were composed of DNA that was rich in the bases guanine and cytosine </li></ul>
  9. 9. G-Banding Technique <ul><li>Giemsa is the most commonly used stain in cytogenetic analysis </li></ul><ul><li>Staining a metaphase chromosome with a Giemsa stain is referred to as G-banding </li></ul><ul><li>Unlike Q-banding, most G-banding techniques require pretreating </li></ul><ul><li>Chromosomes are pretreated with either salt or a proteolytic (protein-digesting) enzyme. </li></ul>
  10. 10. G-Banding Technique (contd..) <ul><li>“ GTG banding” refers to the process in which G-banding is preceded by treating chromosomes with trypsin. </li></ul><ul><li>G-banding preferentially stains the regions of DNA that are rich in adenine and thymine. </li></ul><ul><li>The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light. </li></ul>
  11. 11. G-Banding Technique (contd..) <ul><li>Standard G-band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes. </li></ul><ul><li>With high-resolution G-banding techniques, as many as two thousand different bands have been catalogued on the twenty-four human chromosomes. </li></ul>
  12. 12. G-banded metaphase from a normal female
  13. 13. G-banded karyotype from a normal female.
  14. 14. Yunis’s Technique <ul><li>Jorge Yunis introduced a technique to synchronize cells so they are held at the same stage in the cell cycle. </li></ul><ul><li>Cells are synchronized by making them deficient in folate, thereby inhibiting DNA synthesis. </li></ul><ul><li>By rescuing the cells with thymidine, DNA synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be predicted. </li></ul><ul><li>Yunis’s technique allows more bands to be resolved,as chromosomes produced from either prophase or prometaphase are less condensed and are thus longer than metaphase chromosomes. </li></ul>
  15. 15. R-Banding Technique <ul><li>R-banding is the reverse pattern of G bands </li></ul><ul><li>G-positive bands are light with R-banding method, and vice versa </li></ul><ul><li>R-banding involves pretreating cells with a hot salt solution that denatures DNA that is rich in adenine and thymine </li></ul><ul><li>The chromosomes are then stained with Giemsa. </li></ul><ul><li>R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding. </li></ul>
  16. 16. C-Banding Technique <ul><li>C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA. </li></ul>
  17. 17. NOR-Staining <ul><li>NOR-staining, where NOR is an abbreviation for “nucleolar organizing region,” </li></ul><ul><li>refers to a silver staining method that identifies genes for ribosomal RNA that were active in a previous cell cycle </li></ul>
  18. 18. Fluorescent in situ Hybridization (FISH) <ul><li>FISH is a molecular cytogenetic technique </li></ul><ul><li>Allows cytogeneticists to analyze chromosome resolution at the DNA or gene level </li></ul><ul><li>FISH can be performed on dividing (metaphase) and non-dividing (interphase) cells to identify numerical and structural abnormalities resulting from genetic disorders. </li></ul>
  19. 19. FISH (contd…) <ul><li>Utilizes Probes; there are 3 catogories </li></ul><ul><li>1. Repetitive sequences, including alpha satellite DNA, that bind to the centromere of a chromosome </li></ul><ul><li>2. DNA segments, representative of the entire chromosome, that will bind to and cover the entire length of a particular chromosome </li></ul><ul><li>3.DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified. </li></ul>
  20. 20. FISH (contd…) <ul><li>Probes are “Tagged” with fluorecent nucleotides either directly or indirectly </li></ul><ul><li>This is done by attaching nucleotides to small molecules such as biotin digoxygenin, or dinitrophenyl, to which fluorescent antibodies can later be bound </li></ul><ul><li>The cells are then viewed with a fluorescence microscope. The fluorescent signals represent the probe(s) that is bound to the chromosomes </li></ul>
  21. 21. Chromosome Painting
  22. 22. References: <ul><li>Genetics by Richard Robinson, The Macmillan Science Library, Volume 1; Page 125-129. </li></ul><ul><li>Encylopedia of Molecular Biology, Pg 2347. </li></ul>