Karyotyping techniques, In-situ hybridization and various applications
- 1. COURSE NO. : GPB-505 3(2+1) COURSE TITLE : Principles of cytogenetics LECTURE 6 & 8 : Introduction to techniques of karyotyping. In situ hybridization and various applications. PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY AGRICULTURAL COLLEGE , POLASA, JAGTIAL Submitted by: Ramidi Gayathri JAM/2023-11 M.Sc. Ag First year (Dept. of Genetics and Plant Breeding)
- 2. INTRODUCTION The word karyotyping was derived from the ancient Greek word “Karyon” which means “Kernel”, “seed” or “nucleus”. Chromosomes were first observed in plants thus the name karyotype is given to it. In 1842, Carl Wilhelm von Nageli had observed the plant cell nucleus. He observed a thread-like structure and named it transitory cytoplasts which were actually chromosomes. In 1888, Waldeyer named Nageli’s findings as chromosomes. All of our DNA (except cytoplasmic DNA), are located on chromosomes. Any alteration in numbers or structure of chromosomes causes abnormalities known as genetic disease or genetic abnormality.
- 3. Cytogenetics is the diagnostic study of the structure and properties of chromosomes and cell division, which employs various methods, one of them being "karyotyping.“ It has made it possible to visualize undetected chromosomal anomalies such as small portions of chromosomes and translocations of tiny parts of chromosomes to one another. Because such procedures also enabled each pair of chromosomes to be distinguished individually, it has helped to further understanding the chromosomal basis of certain important genetic disorders.
- 4. "karyotype" refers to the constitution of chromosomes of an individual. Particular chromosome complement of an individual or a related group of individuals, as defined by the chromosome size, morphology and number is known as a Karyotype. “The process of arranging, pairing, and organizing chromosomes to find chromosomal variations is known as karyotyping.” The aim of preparing a karyogram or karyotype is to find out any chromosomal variations. DEFINITION
- 5. Unlike molecular techniques such as PCR, DNA sequencing, or microarray, the cytogenetic techniques are quite tedious, time-consuming, and less effective. Techniques like cell culture, staining, and banding are key elements of cytogenetics and thus are very important in performing karyotyping. Though it is less beneficial than molecular genetic techniques, still, scientists are using karyotyping for screening and diagnosis of various genetic diseases for a long time. It is powerful enough to solve the subtle mysteries of chromosomes and related abnormalities. Change in chromosome number & structure, deletions, duplications and other vast copy number variations can be screened by conventional karyotyping technique.
- 6. Cytogeneticists are using it in clinical medicines to find out trisomies like down syndrome, Patau syndrome and Edward’s syndrome. Interestingly, the technique was originally discovered to study chromosomal constituents of the plant genome not for the human genome but over the period of time, it becomes popular to screen genetic abnormalities.
- 7. Importance of Karyotyping in Plant Studies 1. Genome Characterization Karyotyping allows researchers to identify the unique chromosome complement of a plant species, providing a detailed genetic blueprint that can be used for comparative analysis and taxonomic classification. 2. Cytogenetic Screening By examining chromosomes, scientists can detect chromosomal abnormalities, such as ploidy changes or structural rearrangements, which can have significant impacts on plant growth, development, and performance.
- 8. 3. Breeding and Biotechnology Karyotyping is a crucial tool in plant breeding programs, enabling the identification of desirable genetic traits and the selection of parents for hybridization. It also supports the development of new plant varieties through genetic engineering and other biotechnological approaches.
- 9. STEPS 1. Sample Collection: Common sources include root tips, flower buds, or young leaves because these tissues have actively dividing cells. 2. Pre-Treatment: The collected samples are treated with substances like colchicine or ice water to arrest cell division at the metaphase stage, where chromosomes are most visible and condensed. 3. Fixation: Cells are fixed using chemicals like Carnoy’s solution (a mixture of ethanol and acetic acid) to preserve cell structure and prepare them for staining.
- 10. 4. Staining: Stains like Giemsa, aceto-orcein, or fluorescent dyes are used to make chromosomes visible under a microscope. 5. Microscopy: The stained cells are examined under a microscope, and photographs are taken to capture the chromosome images. 6. Analysis: Chromosomes are paired and arranged according to size, banding pattern, and centromere position to create a karyogram. This involves matching homologous chromosomes and organizing them in a standardized format.
- 11. Staining Methods for Chromosome Visualization Aceto-Carmine Staining This classic staining method involves the use of carmine dye, which binds to the DNA in the chromosomes, creating a reddish-purple color that enhances their visibility under the microscope. Fluorescent Staining Fluorescent dyes, such as DAPI or Giemsa, are commonly used to stain plant chromosomes. These dyes bind to specific DNA sequences, producing a characteristic banding pattern that can be observed under a fluorescence microscope.
- 12. Banding Technique Advanced staining methods, like C-banding and N-banding, can reveal the presence of specific chromosomal structures, such as centromeres and nucleolar organizer regions, further aiding in the identification and characterization of plant chromosomes.
- 13. Techniques used in Plant Karyotyping Classical Karyotyping: Giemsa Staining: This traditional method involves staining chromosomes with Giemsa dye, which binds to DNA and produces a visible banding pattern. It's simple and cost- effective but has lower resolution compared to modern techniques. Procedure: 1. Collect root tips and pre-treat them to arrest cell division. 2. Fix the cells and stain with Giemsa. 3. Prepare microscope slides and observe the stained chromosomes. 4. Photograph the chromosomes and arrange them into a karyogram.
- 14. IN- SITU HYBRIDIZATION In situ Hybridization (ISH) is a powerful method to localize nucleic acid sequences in vivo i.e. in tissues, cells, organelles, nuclei or chromosomes by using appropriate probes. With ISH, nucleic acids are localized in their original or proper place on chromosome will be identified.
- 15. How ? The preparation of biological material that has to be investigated. Probes are labeled. Both probes target nucleic acid are denatured. Single stranded probe gets hybridized to the region where it found sequences complementary to it. Hybridization is detected Hybridization is visualized.
- 16. DIFFERENT TYPES LOCALIZED BY ISH DNA sequences RNA sequences Viral sequences Repetitive seq. Helps to study the spatial & temporal patters of gene expression. Forms the basis of diagnosis of several viral diseases Unique seq. Whole chromosome or a part of chromosome Whole genome
- 17. PROBES Probes are fragments of DNA that were isolated, purified, amplified and labelled with fluorophore, with sites for interaction with antibody or avidin. Probes can vary in length from a few base pairs for synthetic oligonucleotides to larger than one Mbp. Probe size is important because longer probes hybridize more specifically than shorter ones. There are 3 main types of probes for FISH: 1) locus specific probes, 2) centrometric repeat probes -- repeated DNA sequences, 3) whole chromosome probes Fluorescent dyes- Cy 5 (far red), Cy 3 (orange), FLUOS (green)
- 18. FISH AND GISH Fluorescence in situ hybridization (FISH) : A fluorescent molecule is deposited at the site of in situ hybridization location of genes or DNA can be visualized on chromosomes. Genomic in situ hybridization (GISH) : Total genomic DNA is used as probe in hybridization experiments
- 19. FLUORESCENCE IN SITU HYBRIDIZATION (FISH) Is a cytogenetic technique that allows detection and localization of specific nucleic acid sequences on morphologically preserved chromosomes. It uses florescent probes that bind only to those parts of chromosomes which show a high degree of sequence similarity. Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination.
- 21. HISTORY (1969)- In situ hybridization technique was developed by joseph G Gall and Mary lou Pardue and John et al.(1969) (1985)- The non isotopic in situ hybridization using biotin labeled DNA probes was first introduced in plant species by Rayburn and Gill (1991)- The first application of FISH to plant cytogenetics was the work of Leitch et al.(1991)
- 22. APPLICATIONS Can visualize specific cytogenetic abnormalities (copy number aberrations) • chromosomal deletion, amplification, translocation Each fluorescently labeled probe that hybridizes to a cell nucleus in the tissue of interest will appear as a distinct fluorescent dot • Diploid nuclei will have two dots • If there is duplication in the region of interest, the gain will result in more than two dots. • If there is a loss in the region of interest, one or zero dot will result. • Was often used during Metaphase but is now used on Interphase chromosomes as well. Advantage : Less labor-intensive method for confirming the presence of a DNA segment within an entire genome than other conventional methods like Southern blotting
- 23. PROCEDURE 1. Prepare chromosome spreads on slides. 2. Denature the DNA on the slides to make it single-stranded. 3. Apply fluorescently labeled DNA probes to the slides. 4. Hybridize the probes to the target DNA sequences. 5. Wash off excess probes and visualize under a fluorescence microscope.
- 24. GENOMIC IN SITU HYBRIDIZATION (GISH) Genomic In-situ hybridization(GISH) is a cytogenetic technique that allows the detection and localization of specific nucleic acid sequences on morphologically preserved chromosome using genomic DNA of donor species as prob.
- 25. INTRODUCTION GISH is quick, accurate, sensitive, informative and a comparative approach. GISH technique is an advancement in the fluorescence in situ hybridization (FISH) technique.
- 26. HISTORY GISH for plants was developed inn 1987 by M.D. Bennett. GISH was mainly developed for the animal hybrid cell in 1986 In 1987, the Plant Breeding institute Cambridge was used this technique in plants M.D Bennett
- 27. PRINCIPLE This technique involves the extraction of labeling DNA of one organism and to use as a probe to target the genome of another organism. The part of genome that are similar to the probe hybridize to from a probe target complex
- 28. PROCEDURE Take probe DNA Blocking DNA fragmentation Preparation of slide Denaturation of probe and blocking DNA in the hybridization mixture Addition of probe and blocking DNA with the hybridization mixture Chromosome DNA denaturation Hybridization of blocking DNA and probe in the target sequence of the chromosome Detection of the probe in the chromosome of one parent Chromosome DNA molecule of thee second parent related to the unlabeled blocking DNA
- 31. USES It is used to identify chromosomal rearrangements in cancer patients. Chromosomal identification in cell. Detect the specific nucleotide sequence within cell and tissues. Unique point among the studies of cell, biology, cytogenetics and molecular genetics It is possible to detect single copy sequence on chromosome with probes. genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics It will useful for investigating the origins of wild and cultivated polyploid plant species
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