Cytogenetic Methods PDF

Cytogenetic method Dr. Alvita Vilkevičiūtė, LSMU BSGTI [email protected] Kaunas, 2024  Cytogenetics Cytogenetics is a branch of genetic science that studies the structure, functions and pathology of chromosomes; Chromosome - the part of the ce...

Cytogenetic method Dr. Alvita Vilkevičiūtė, LSMU BSGTI [email protected] Kaunas, 2024  Cytogenetics Cytogenetics is a branch of genetic science that studies the structure, functions and pathology of chromosomes; Chromosome - the part of the cell nucleus that consists of DNA and histones. 2 DNA organization in the nucleus Almost all DNA in eukaryotes is concentrated in the nucleus. Together with proteins, it forms a unique dynamic structure - chromatin; Eukaryotic chromatin is a complex nucleoprotein composed of: DNA, Histone proteins; Non-histone proteins; RNA (< 10% of DNA mass). 3 Chromatin Table 1. Comparison of properties of euchromatin and heterochromatin Properties of Chromatin Euchromatin Heterochromatin Structure Less condensed, open, Condensed, closed, accessible inaccessible DNA sequences Coding genes Repeating elements Activity Transcriptionally active Transcriptionally inactive 4 Nucleosome is a structural and regulatory unit of chromatin The nucleosome is formed by histone proteins around which the DNA revolves; One nucleosome consists of: Histone octamer (two molecules each of four types of histones: H2A, H2B, H3 and H4); The histone octamer binds to approximately 200 nucleotide pairs of DNA; One linker histone (eg, H1) molecule is bound to one nucleosome in chromatin; The first level of chromatin organization. Figure 1. Structural components of the nucleosome 5 Levels of chromatin structural organization Doublestrand DNA 10 nm 10 nm chromatin fiber 30 nm chromatin fiber Chromatin loops Condensed chromatin Figure 2. Levels of chromatin structural organization Chromosome 6 Chromosome structure Figure 3. Chromosome structure 7 Chromosome types Figure 4. Chromosome types based ont the centromere location 8 Chromosomal numbering system 6p24.32 6p - chromosome short arm 2 - region 4 – sub region (band) 3 – sub-band 2 – sub-sub band Figure 5. Chromosomal numbering system 9 Karyotype Karyotype - an organized set of chromosomes according to size and shape; Each pair of chromosomes is marked with numbers, except for the sex ones. Figure 6. Human karyotype 10 Chromosome ideograms Ideogram – a graphic layout of a chromosome, which indicates the positions of chromosome structures (shoulders, centromeres, telomeres) and coding genes; According to the staining (banding) method and the stage of cell division, chromosomal segments of different resolution are obtained, according to which the exact location of the gene or chromosomal aberration can be indicated Figure7. Different resolution ideograms of human chromosome 7. 11 Use of cytogenetic testing When studying changes in the number and structure of chromosomes in breeds, lines, families; Studying the relationship between chromosomal changes and reproductive characteristics, productivity, vitality, disease resistance of animals; For the search of chromosomal markers; During pre-implantation and prenatal diagnostics; When determining the sex of embryos and chromosomal changes; Evaluating the effect of mitogens on the genome; By making chromosomal maps. 12 Variation in chromosome sets between species Figure 8. Sets of chromosomes of different organisms 13 Human karyotype (I) Figure 9. Human karyotype 14  Human karyotype (II) Table 2. Human chromosome groups Grupė Chromosomos Apibūdinimas A 1, 2, 3 Large-sized metacentric B 4, 5 Large submetacentric C 6, 7 8, 9, 10, 11, 12 Medium-sized submetacentric ir X D 13, 14, 15 Medium-sized acrocentric and has satelites E 16, 17, 18 16 - Medium-sized metacentric, 17 ir 18 - Medium- sized submetacentric F 19, 20 Very small metacentric G 21, 22, Y 21 and 22 – very small acrocentric and has satelites, Y – very small acrocentric and without satelites 15 A B Figure 10. A – Horse karyotype; B – Sheep karyotype 16 A B Figure 11. A – Domestic cat karyotype; B – Male cattle (Bull) karyotype 17 Chromosomal abnormalities Changes in the number of chromosomes: Polyploidy is a regular increase in the total set of chromosomes (3n, 4n). Often formed as a result of interspecific crossbreeding; Aneuploidy is a change in the number of individual chromosomes: Trisomy (2n+1) – when an individual has three chromosomes instead of a pair; Monosomy (2n-1) – when an individual has one chromosome instead of a pair. 18  Polyploidy A B Figure 12. A – Triploid catttle karyotype (90, XXX); B – Triploid human karyotype (69, XXY) 19  Aneuploidy A B Figure 13. A – Mare (female horse) X chromosome trisomy; B – Canine X chromosome monosomy 20  Inheritance of aneuploidy A B Figure 14. A - Nondisjunction of sister chromatids in meiosis II. B - Nondisjunction of homologous chromosomes in meiosis I. 21 Chromosomal abnormalities Changes in the structure of chromosomes: Deletion - loss of a chromosome segment; Gap - damage to the protein coat of the chromosome without breaking the DNA; Translocation - when a segment from one chromosome is inserted into another chromosome; fusion of two chromosomes in the region of centromeres (Robertson translocation). Duplication - repetition of the same chromosome segment two or more times; Inversion - a 180-degree turn of a chromosome segment; Insertion - a change in the location of a segment in the same chromosome; 22  Deletion and gap A deletion B gap X chromosome Y chromosome Figure 19. A – A gap in the cattle (female) autosome; B – A deletion in the cow (male) autosome 23  Translocation A B X chromosome Robertsonian translocation Y chromosome Figure 20. A – Translocation; B –Robertsonian translocation in the bull genome. 24 Inheritance of chromosomes with translocation P: 46,XX x 46, XY, t (7;18)(q35;q12) P: x G: ; ; ; ; 7 7 18 18 7 7 18 18 7 18 7 18 7 18 7 18 7 18 7 18 del(18)(q12),dup(7)(q35) 7 18 7 7 18 18 Normal 7 18 7 7 18 18 t (7;18)(q35;q12) 7 18 7 7 18 18 del(7)(q35),dup(18)(q12) 7 18 7 7 18 18 25 Deletion and duplication A B Figure 21. A – Deletion model; B - Duplication model. 26 Inversion A B Figure 22. A – Paracentric inversion; B – Pericentric inversion 27 Changes in the number and structure of chromosomes Cytogenetics Molecular cytogenetics Giemza staining FISH NOR staining CGH G-banding SKY C-banding R-banding T-banding 28 Methods of preparation of chromosome preparations Direct method - preparations of chromosomes are prepared from tissues (bone marrow, lymph nodes, spleen) in which cell division takes place intensively and a large amount of mitoses is found, even without the use of a substance promoting cell division; Indirect method - preparations of chromosomes are prepared from peripheral blood, using a substance that promotes cell division - a mitogen. 29 GIEMZA staining Metaphase plates are stained with GIEMZA dye; All chromatin is evenly stained pink; It is used for the analysis of the number of chromosomes and large structural changes. Figure 26. Cattle metaphase plate stained with GIEMZA dye 30 G banding The chromosomes are exposed to trypsin, which removes part of the chromosomal proteins, so that the stained chromosomes acquire bands; Bands are specific to the karyotypes of different species of plants and animals, as well as to each pair of a set of chromosomes, so it can be used to recognize homologous chromosomes, identify chromosomal aberrations (deletions, inversions, insertions, translocations); Chromosome preparations are first incubated in trypsin solution, then stained with GIEMZA or acridine orange dye. 31  G banding A- and T-rich sequences are colored dark; C- and G-rich sequences are colored light. Figure 27. Human karyotype. Chromosomes are stained with the G method 32  R banding Reverse G method: sequences rich in T and A are colored light, G and C risch parts are dark; Chromosome preparations are first incubated in 85ºC phosphate buffer, then stained with GIEMZA or acridine orange. Figure 28. Comparison of G and R staining methods 33 C banding The staining process involves the denaturation and renaturation of DNA by sequential acid and alkali treatment; The structural heterochromatin located near the centromeres rapidly renaturates and is stained dark (“C” blocks), while the rest of the chromosome is light; "C" staining is used to identify sex chromosomes and to study chromosomal aberrations, especially translocations. Figure 29. Human karyotype. Chromosomes are stained with C banding 34 34 T banding R staining modification; The telomeric regions of chromosomes are stained; The method is applied to the analysis of abnormalities in telomeric regions. Figure 30. Chromosomes are stained with the T banding method 35 NOR (Nucleolar Organizing Region) staining Helps to identify satellites located on the short arms of acrocentric chromosomes, where there are many RNA genes; Chromosomes are stained with silver nitrate, which specifically binds to active regions of ribosomal DNA. Figure 31. Human karyotype. Chromosomes are stained by NOR method 36 Fluorescence in situ hybridization (FISH) (I) Specific chromosome locations are visualized; Probes labeled with fluorescent signals are used, which bind to the DNA-target region according to the principle of complementarity; Used to detect changes in the number and structure of chromosomes. 37 Fluorescence in situ hybridization (FISH) (II) Process flow: A preparation of chromosomes is prepared or cells are fixed on a slide; DNA is denatured by heat or chemicals; Hybridization in progress; Non-hybridized probes are washed away; The results are analyzed using a fluorescence microscope. Figure 32. FISH method workflow 38 Fluorescence in situ hybridization (FISH) (III) A B Figure 33. Application of the FISH method for the detection of A-structural changes in chromosomes (translocation between chromosomes 8 (red) and 21 (green); B - to detect changes in the number of chromosomes (chromosome 21 (green) trisomy. 39 Spectral Karyotyping (SKY) Figure 34. "Painting" of chromosomes. The human genome is analyzed. 40 Comparative genomic hybridization (CGH) array A change in the chromosome copy number (deletions, duplications, insertions) is determined; No cell culture is required; The method is based on the hybridization of single-stranded DNA fragments; The results are evaluated according to the intensity of the fluorescent signal. 41  CGH array method Process: DNA isolation; DNA staining with fluorochromes; DNA denaturation; Mixing of test and reference DNA; Hybridization with single-stranded DNA fragments in array wells; not bound fragments are washed then; Evaluation of the results - the fluorescent signal is recorded. Figure 35. Diagram of an array-based CGH 42  CGH array results evaluation A Test Reference B DNA DNA Figure 36. CGH results evaluation. A – fluorescence signal explanation B – results displayed on automated analyzer 43  Cytogenetic method Dr. Alvita Vilkevičiūtė, LSMU BSGTI [email protected] Kaunas, 2024