chapter 5. xms banding and karyotyping.pptx

Xms banding and karyotyping in genetics

1. Chapter Five Karyotyping and Major types of chromosome banding

2. Karyotype, Karyotyping and preparation of Ideogram What is karyotype? All species are characterized by a set of chromosomes to carry their genetic information The chromosomal composition of each species has a number of characteristics The karyotype is a set of characteristics that identifies and describes a particular set of set of chromosomes These characteristics include: a. the chromosome number b. relative size of different chromosomes c. position of centromeres and length of chromosome arms d. presence of secondary constrictions and satellites e. banding pattern of the chromosomes f. features of sex chromosomes

3. What is karyotyping? The process of preparation of the karyotype of a species is called karyotyping Karyotyping is now commonly used in clinical diagnosis and clinical genetics Karyotype is prepared from the micrograph of metaphase chromosomes The metaphase chromosome is selected because at this stage the chromosomes will have maximum condensation(thickness),they will be visible through an ordinary laboratory microscope In a karyotype, the choromosomes of an organism are ordered in a series of decreasing size In the case of humans autosomes are numbered from 1 to 22 and arranged in the order of decreasing size Sex chromosomes are arranged after the autosomes

4. What is Ideogram? Ideograms are a schematic representation of chromosomes. They show the relative size of the chromosomes and their banding patterns. A banding pattern appears when a tightly coiled chromosome is stained with specific chemical solutions and then viewed under a microscope. Some parts of the chromosome are stained (G-bands) while others refuse to adopt the dye (R-bands). The resulting alternating stained parts form a characteristic banding pattern which can be used to identify a chromosome. The bands can also be used to describe the location of genes or interspersed elements on a chromosome.

5. Human chromosome ideogram 6

6. Major types of chromosome banding Different chromosomes often have similar sizes and centromeric locations, cytogeneticists must use additional methods to accurately identify each type of chromosome within a karyotype . For detailed identification, chromosomes are treated with stains to produce characteristic banding patterns Cytogeneticists use several different staining procedures to identify specific chromosomes

7. Banding Patterns Reveal the Structural Details of Chromosomes To make analysis more efficient, cytologists have developed stains that bind with DNA and generate characteristic banding patterns for different chromosomes Prior to the development of these banding techniques, distinguishing chromosomes from one another was very difficult chromosomes were simply grouped according to their size and the placement of their centromeres 8

8. Cont… The banding pattern of eukaryotic chromosomes is useful in two ways : Individual chromosomes can be distinguished from each other if they have similar sizes and centromeric locations. banding patterns are used to detect if there is a changes in chromosome structure which is difficult to see by using only centromeric orientation & size the positions and sizes of the chromosome bands are constant and specific to the individual chromosome with in species. Therefore , structural rearrangements could be seen by observing the loss/gain of chromosome segments with different staining characteristics

9. Cont ----- Chromosomes can be readily identified at the cytological level by a number of specific staining procedures Comparison of these band can help to match homologous pairs in a karyotype as homologous chromosomes have the same banding pattern Based on the stains used the following banding techniques exist: • G-banding: Giemsa staining • R-banding: (reverse) modified Giemsa staining • C-banding: centromere specific staining • Q-banding: quinacrin staining ( fluorescent)

10. Depending on the staining technique used, such segments may appear as: dark staining, light staining or as brightly fluorescing bands in the chromosomes/ pale fluorescing bands.

11. Q-banding In 1970, Caspersson and his colleagues described the first banding technique, known as Q-banding Q-banding involves staining of chromosomes with a fluorescent dye quinacrine , and patterns are observed by placing the sample under a special type of ultraviolet light microscope ( fluorescence microscopy ) The chromosomes show in a specific pattern of bright fluorescent and dim bands, the bright Q bands corresponding almost exactly to the dark bands seen after G banding.

12. Q-banding The molecular causes for staining differences along the length of a chromosome are complex and it include the base composition of the DNA and local differences in chromatin structure. Q-banding is the first method to be used to identify all 46 human chromosomes However, visualization of the fluorescence pattern requires fluorescence microscope which is very expensive Since then, a variety of other chromosome banding techniques have been developed which use light microscope

13. G banding Today, most karyotypes are stained with Giemsa dye, which have advantages like can be analyzed with ordinary bright-field microscope Better resolution of individual bands, produces a more stable preparation

14. G banding In G-banding, metaphase chromosomes are first treated briefly with trypsin , an enzyme that degrades proteins, before the chromosomes are stained with Giemsa Trypsin partially digests some of the chromosomal proteins, thereby relaxing the chromatin structure and allowing the Giemsa dye access to the DNA The difference between dark- and light-staining regions is chromatin packing density : The G-dark regions are packed more densely, with tighter coils. Thus there is a higher density of DNA to take up the stain.

15. G banding In general, heterochromatic regions, which tend to be AT-rich DNA and relatively gene-poor, stain more darkly in G-banding In contrast, less condensed euchromatin which tends to be GC-rich and more transcriptionally active incorporates less Giemsa stain, and these regions appear as light bands in G-banding In the complete set of 23 human chromosomes, there are approximately 850 G-dark bands visible during a stage of mitosis G-banding produces reproducible patterns for each chromosome, and these patterns are shared between the individuals of a species.

16. E.g. Giemsa -stained human chromosomes, as they appear under a microscope dhc 17

17. R-banding R-banding also involves Giemsa stain, but the procedure generates the reverse pattern from G-banding. In R-banding, the chromosomes are heated before Giemsa stain is applied. R-banding is often used to provide critical details about gene-rich regions that are located near the telomeres . Especially when regions that stain poorly by G or Q banding are examined, R banding gives a pattern that is easier to analyze than that given by G or Q banding

18. R-banding Since it stains gene-rich chromatin, it could be used to visualize small structural rearrangements in the parts of the genome that are most likely to result in phenotypic abnormalities R-bands are most useful in identifying abnormalities involving the terminal regions of chromosomes, which are lighter staining by G- and or Q-banding.

19. C-Banding Noncoding constitutive heterochromatin, such as the repetitive DNA surrounding the centromeres of all of the chromosomes , Those DNA sequences around the centromere replicates later in the cell cycle than other chromatin and exhibits special characteristics of stability under extreme conditions of heat and chemical exposure This property can be exploited to produce a unique banding pattern (C-banding) -stains darkly and all other chromatin remains pale C-Banding is produced by treatment of chromatin with acidic and then basic solutions followed by staining with Giemsa C-banding is of limited use in the clinical laboratory but used for the study of chromosomal polymorphisms in plant population

20. Ap Applications of karyotyping plications of karyotyping A karyotype allows us to determine the chromosome makeup of an individual. To detect chromosome number variants It can show numerical chromosomal variations that can cause a range of disorders in humans. To detect Chromosome morphological aberrations Morphological aberrations may disturb, or even prevent, recombination and they may cause sterility. Extra, missing, or abnormal positions of chromosome pieces can cause problems with a person’s growth, development, and body functions . For prenatal sex determination

21. To map location of genes on a chromosome Used for mapping the locations of genes based on abnormalities that occur due to absence of a chromosome or segment of a chromosome. In species descriptions ( cytotaxonomy ) Even simple chromosome number differences could well be a reason to separate different forms into distinct species Give information to the breeder regarding barriers to the crossing plants from related or more distant species . The karyotype can indicate primitive and advanced features of an organism

22. To study evolutionary relationship between species Comparisons of chromosome banding patterns can confirm evolutionary relationships between species and also reveal changes in karyotype that may have been important in speciation. Sometimes , the chromosome number or even the number of genomes per nucleus are accepted as the maximum of information comparing the ploidy level of related species The number of genomes determines the size of the nucleus and indirectly the size of the cell

23. Preparing Karyotypes from Mitotic Cells with human examples Karyotypes are prepared from mitotic cells that have been arrested in the metaphase stage of the cell cycle when chromosomes assume their most condensed conformations All of the organs (heart, liver, pancreas, blood etc .) in our body are made up of cells. Each cell contains the genetic information . Therefore, A variety of tissue types can be used as a source of these cells. 25

24. For prenatal diagnosis, amniotic fluid or chorionic villus specimens are used as the source of cells . For other diagnoses, karyotypes are often generated from peripheral blood specimens or a skin biopsy. Bone marrow or tumor biopsy samples to diagnose certain cancers or evaluate their course and the effectiveness of treatment

25. Amniosynthesis or chorionic villus sampling Amniotic fluid or chorionic villus sampling to detect chromosomal disorders in the fetus. Fetal karyotypes can help determine chromosomal abnormalities before a baby is even born It is normally considered in situations in which parents are at risk of having an infant with a genetic disorder when: Parents have had a previous child with chromosomal abnormalities Higher maternal age

26. Amniocentesis Amniocentesis: is sampling of fetal cells from amniotic fluid (cells) During this procedure the fluid, which surrounds the baby and provides protection, is taken from the amniotic sac by a long needle inserted into the woman’s abdomen

27. Chorionic Villus Sampling It is more specialized than amniocentesis and can be done earlier in pregnancy (during about 10-12 weeks of pregnancy). A sample is taken from the chorionic villus, the fetal tissue that forms part of the placenta, by suction. In this procedure, a small, flexible catheter is inserted through the vagina or abdomen into the uterus and is guided by ultrasound images. In general, blood samples give the best quality chromosomes and therefore provide the best chance of detecting small subtle chromosome abnormalities.

28. Blood sample to evaluate the cause of birth defects, to evaluate couples with chromosomally abnormal children. to detect the cause of infertility or repeated miscarriages. to evaluate women who aren't menstruating to examine abnormal sexual development, particularly when there is doubt about true gender.

29. Video ( https:// www.youtube.com/watch?v=7ShPzzrCetE ) 31

30. The process of generating a karyotype begins with the short-term culture of cells derived from a specimen . After a period of cell growth and multiplication, dividing cells are arrested in metaphase by addition of colchicine prevents assembly of the mitotic spindle The cells are next treated with a hypotonic solution that causes their nuclei to swell and the cells to burst The nuclei are then treated with a chemical fixative, dropped on a glass slide, and treated with various stains that reveal structural features of the chromosomes

31. The chromosome spreads are then photographed and the individual chromosomes cut out The chromosomes are then matched with their homologues and are aligned on the karyotype form

32. Presenting the Karyotype There are two main ways to present a karyotype graphically . The first is the karyogram , in which a representative cell is photographed The chromosomes cut out from the photograph and collected in pairs of (presumed) homologues, and the pairs lined up in descending order of length 34

33. In a karyotype, the chromosomes may look bent or twisted. This is normal and is a result of how they were sitting on the slide when the photograph was taken It is a very demonstrative way, but when the chromosomes do not have specific morphological characteristics that make individual identification possible, it may be misleading Within a karyogram, chromosomes are aligned along a horizontal axis shared by their centromeres 35

34. Standard terminology is used for naming band and gene locations in chromosomes The pattern of bands on each chromosome is numbered on each arm from the centromere to the telomere By use of this numbering system, the location of any particular band as well as the DNA sequences and genes within it and its involvement in a chromosomal abnormality can be described precisely and unambiguously 36

35. First , the autosomes are numbered and arranged based on size from longest to shortest E.g for humans it range from 1-22, Then chromosomes that determine sex are labeled and put at the end regardless of their size (e.g. X or Y)

36. Then, the chromosome is divided into the p and q arm regions. The short arm is designated "p", and the long arm "q". By convention the “p” arms are placed at the top and “q” at the bottom Each arm is then divided into major sections /regions and subsections/bands that are numbered consecutively out from the centromere Each region divided into bands identified with a number Example - 1q2.4 The first chromosome, long arm, second region of the chromosome, the fourth band of that sub-region

37. The other alternative representation of the karyotype is the idiogram, an ordered set of idealized chromosome diagrams with : The length representing the length of each chromosome (the relative length i.e., percentage of total genome length) The place of the primary and secondary constrictions, Other recognizable markers like C-bands, drawn at the proper locations.

38. Human chromosome ideogram 40

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40. Human chromosome ideograms in genetic disease diagnosis 42