Karyotyping
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This document discusses karyotyping and its applications. It begins with an overview of cytogenetics and the history of karyotyping. It then describes the different types of karyotyping including classic and digital karyotyping. The methodology section explains the process of karyotyping including chromosome staining techniques like Q-, G-, N-, and C-banding. Finally, the applications of karyotyping are discussed, such as detecting chromosomal abnormalities, aneuploidies, and genetic disorders. Karyotyping provides a genome-wide view of an individual's chromosomes and is useful for clinical diagnosis and research.
Karyotyping
- 1. Karyotyping Zahra Naz Athar Hussain Waqas Ahmad By Mphil-Biotechnology (2nd semester) National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan
- 2. • Cytogenetics • History • Karyotyping • Types • Methodology • Staining techniques • Applications • Conclusion 6/18/2017 2
- 3. Cytogenetics “Study of the structure and properties of chromosomes, chromosomal behaviour during mitosis and meiosis, chromosomal influence on the phenotype and the factors that cause chromosomal changes” Rieger, R.; Michaelis, A.; Green, M.M. (1968), A glossary of genetics and cytogenetics: Classical and molecular, New York: Springer-Verlag 6/18/2017 3
- 4. History of Cytogenetics 1835 Hugo Von Mohl described cell division 1888 Von Waldeyer gave name chromosome 20th century- Golden era of genetics 1903 Johannsen coined the terms gene, genotype and phenotype 1982 Bloom and Carbon revealed molecular structure of centromere Ferguson-Smith, M. A. (2015). History and evolution of cytogenetics. Molecular Cytogenetics 8, 19.6/18/2017 4
- 5. Karyotyping (A cytogenetic technique) “Karyotyping is the process of pairing and ordering all the chromosomes of an organism, thus providing a genome wide snapshot of an individual's chromosomes (karyogram/ Idiogram)” https://www.nature.com/scitable/topicpage/karyotyping-for-chromosomal-abnormalities-298 6/18/2017 5
- 7. Classic karyotype Cytogenetics • Chromosome number and arrangement studies • Smaller chromosomal aberrations are detected e.g. translocations • Complex chromosomal rearrangements often remain hidden Riegel, M. (2014). Human molecular cytogenetics: From cells to nucleotides. Genet. Mol. Biol. 37, 194-209. 6/18/2017 7
- 8. Digital karyotyping • Based on quantitative analysis of DNA (DNA copy number) • Detection by using short sequence tags (SST) (21 base pairs usually) Wang, T.-L., Maierhofer, C., Speicher, M. R., Lengauer, C., Vogelstein, B., Kinzler, K. W., and Velculescu, V. E. (2002). Digital karyotyping. Proceedings of the National Academy of Sciences of the United States of America 99, 16156. 6/18/2017 8 Chronic lymphocytic leukemia sample
- 9. Methodology Redrawn: Image from https://infograph.venngage.com/p/219813/karyotype
- 11. 1. On basis of size www.shutterstock.com.279849302 6/18/2017 11
- 12. 2. On basis of banding http://www.evolutionrevolution.eu/es/ 6/18/2017 12
- 13. 3. On basis of position of centromere http://learn.genetics.utah.edu/content/basics/readchromosomes/ Group A: Large metacentric Group B: Large submetacentric Group C: Medium submetacentric (X) Group D: Medium acrocentric Group E: Short submetacentric Group F: Short metacentric Group G: Short acrocentric (Y) 6/18/2017 13
- 17. Introduction
- 18. Classification of Banding Techniques Based on GC and AT rich regions. Constitutive Heterochromatin Region. Always metaphase chromosomes whose size has condensed and whose diameter is increased are used for chromosome banding studies after fixing stage. Wendy, A. B.,2001. Karyotype analysis and chromosome banding. Nature, 1-6.
- 19. The banding techniques fall into two principal groups: • Bands, distributed along the length of the whole chromosome, such as G-, Q-band • Those that stain a number of specific bands.These include methods which reveal centromeric bands, C-bands, and nucleolus organizer regions, NOR's (at terminal regions of chromosomes). Principle Moore, C. M. and Best, R. G., 2002. Chromosome preparation and banding. Nature, 1-6.
- 20. Banding Techniques Q (Quinarcine) G (Giemsa) N (NOR) C (Centromeric) 1958 1971 1973 1978 Casperson et.al Summer et.al Matsui & Sasaki Linde &Laursen
- 21. 1.Q Banding • Quinacrine mustard, an alkylating agent, was the first chemical to band chromosomes viewed under a fluorescence microscope. • Dark and light florescence Ried, T., et.al ,1998. Chromosome painting : a useful art. Human Molecular Genetics, Vol(7),1619- 1626.
- 22. 1.Q Banding Techniques Advantages • Simple and Versatile. • Used where G band is not accepted. • Used in study of chromosome heteromorphism. Disadvantages • Tendency to fade during examination. Photo-degradation . UV light breaks the chemical bond.
- 23. • It stain regions rich in adenine and thymine. • Standard G band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes. 2.G Banding • Methylene Blue • Eosin • Azure B Giemsa solution https://en.wikipedia.org/wiki/Giemsa_stain
- 24. 2.G Banding Techniques Chromosome Weak Trypsin / urea/ protease Treated with Giemsa Banding pattern To denature protein Interaction of the DNA with thiazine & eosin components of stain with Sulfar regions of DNA Methylene Azure+ Methylene Violet+ Methylene Blue+ Eosine http://www.protocol-online.org/biology-forums/posts/15995.html
- 25. G Banding Techniques Advantages • Used in identification of bands rich in Sulphur content. • Used in the identification of chromosomal abnormalities • Gene Mapping. Disadvantages • Not used in plants. • Required Pre-treatment by Salt or proteolytic enzyme
- 26. G banding not used in Plants. Why???? • Human mitotic metaphase chromosome is 2.3 times shorter. • Plant mitotic metaphase chromosome is 10 times more shorter than human chromosome. • Hence difficult to demonstrate the arrangement of bands at this level of saturation with G banding technique. Source: Greilhuber, J (1977). Why Plant chromosome do not show G bands? .Theory of Applied Genetics, Vol 50(3): 121- 124.
- 27. • NOR-banding involves silver staining (silver nitrate solution) of the "nucleolar organizing region", which contains rRNA genes. • NOR region is highly condensed. • It can neither be expressed to nor encode any protein 3. N(NOR) Banding
- 28. N Banding Techniques Advantages • Used in the identification of nucleolar organizer region. • Superior banding pattern for plants. Disadvantages Time consuming both in technique and reagent preparation.
- 29. C (centromeric)-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA. 4. C Banding
- 30. C Banding Techniques Advantages • Identification of chromosomes particularly in insects and plants. • Identification of centromere position. • Gene mapping. Disadvantages • C-banding methods do not permit identification of every chromosome in the somatic cell complement Gupta ,P. K., 2012. Cytogenetics an advanced study, Chapter 1: 3-16.
- 32. APPLICATIONS OF KARYOTYPING • Detection of chromosomal aberration • Identify loss and addition of chromosome • Determine the risk of individual • Detect aneuploidy • Prebirth diagnosis of genetic diseases • Gender identification http://www.biotecharticles.com/Biology-Article/Karyotype-and-Its-Applications-in-Human-Health-2014.html
- 33. Diagnostic of different genetic Disorder in human “Genetic disorder is a disease that is caused by an abnormality in an individual's DNA”. A. Autosomal Disorder Autosomal Dominant Autosomal Recessive B. Allosomal Disorder X- linked dominant X- linked recessive Y- linked 3. Mitochondrial Disorder https://www.genome.gov/10001204/specific-genetic-disorders/
- 34. Mitochondrial disorder • It is a disorder caused by mutations (or changes) in either mitochondrial DNA or nuclear DNA • These are only passed on from mother. • 1:4000 https://www.genome.gov/10001204/specific-genetic-disorders/
- 35. Trisomy • Three instances of a particular chromosome, instead of the normal two. • Aged mothers • High risk of aneuploidy chromosome • Fertilization of an egg having aneuploidy chromosome result in fetus with syndrome. • Trisomy 21 (Down’s syndrome) • Trisomy 18 (Edwards syndrome) • Trisomy 13 (Patau syndrome) https://www.genome.gov/10001204/specific-genetic-disorders/
- 36. Down Syndrome • Down Syndrome also known as Trisomy 21, is a chromosomal condition caused by the presence of all or part of an extra 21st chromosome. • Non-disjunction during meiosis • John Langdon Down (1866) • Short height • Severe mental deficiency with decline in the IQ with age • Average IQ:50 • Brachycephaly with flat face and occiput • Flat and low nasal bridge https://www.genome.gov/10001204/specific-genetic-disorders/
- 37. Edward Syndrome • Edward’s Syndrome also known as Trisomy 18 (T18) or Trisomy E. • 2nd most common disorder after down syndrome • Mental deficiency • Growth retardation • Prominent occiput with elongated head • Webbing of the neck • Short sternum • Micrognathia http://syndrome.org/patau-syndrome/
- 38. Patau Syndrome • A syndrome in which a patient had an additional chromosome on position 13 due to non-disjunction of chromosomes during meiosis • Patau syndrome 1 in 10,000 . • Mental deficiency, Low birth weight, Abnormal development of frontal lobe ,Hypoplasia of cerebellum etc http://emedicine.medscape.com/article/947706-overview
- 39. Autosomal deletion • An abnormality of DNA that involves missing material. • These can range from very small to very large • The most common types of autosomal deletion syndromes are: • Wolf-Hirschhorn Syndrome • Cri Du Chat https://link.springer.com/chapter/10.1007/978-1-4613-0139-4_15
- 40. Autosomal deletion 2. CRI DU CHAT(5p deletion syndrome): • Jerome Lejeune in 1963 • DELETION on the short arm of chromosome 5. • Common in female • Cries like a CAT 1. Wolf-Hirschhorn Syndrome (Pitt Syndrome ): • Herbert L. Cooper(1961) • DELETION of the short arm of chromosome 4 https://link.springer.com/chapter/10.1007/978-1-4613-0139-4_15
- 41. Allosomal disorder Turner syndrome 1 X0=45 2 Extra skin on NECK 3 Infertile 4 Shorter than average 5 heart defects and kidney problems https://ghr.nlm.nih.gov/condition/klinefelter-syndrome
- 42. Kleinfelter’s syndrome 1. XXY=47 2. A shortage of testosterone 3. Gynecomastia 4. reduced facial and body hair 5. Infertile 6/18/2017 42
- 44. 6/18/2017 44
- 45. 6/18/2017 45 Q. & A.
Editor's Notes
- #23: Versatile: Adoptable. To be adopted Heteromarphism : the quality or condition of existing in various forms
- #24: It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. When chromosomes are pre-treated with the proteolytic enzyme trypsin the process is called GTG banding
- #28: Silver nitrat form insoluble with phosphat Ions.. it forms black elementary silver