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Contents
Chapter 1
Cytogenetics and Chromosomics......................................................... 1
Chapter 2
Banding Cytogenetics .......................................................................... 7
Chapter 3
Generation of Microdissection-Derived Painting Probes from
Single Copy Chromosomes ................................................................ 27
Chapter 4
FISH—An Overview.......................................................................... 35
Chapter 5
FISH—Microscopy and Evaluation ................................................... 43
Chapter 6
FISH—in Routine Diagnostic Settings .............................................. 49
Chapter 7
FISH—in Leukemia Diagnostics....................................................... 71
Chapter 8
FISH—in Tissues ............................................................................. 105
Chapter 9
FISH—in Human Sperm and Infertility .......................................... 111
vii
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viii
Contents
Chapter 10 FISH—in Spontaneously Aborted Products of Conception ............ 117
Chapter 11 FISH—Characterization of Chromosomal Alterations,
Recombination, and Outcomes after Segregation............................ 121
Chapter 12 Multicolor-FISH—Methods and Applications................................. 151
Chapter 13 FISH—Centromere- and Heterochromatin-Specifc
Multicolor Probe Sets....................................................................... 157
Chapter 14 FISH—Detection of Individual Radio Sensitivity ........................... 163
Chapter 15 FISH—Detection of CNVs .............................................................. 171
Chapter 16 FISH—Interphase Applications Including Detection of
Chromosome Instability (CIN) ........................................................ 181
Chapter 17 FISH—Determination of Telomere Length
(Q-FISH/CO-FISH).......................................................................... 189
Chapter 18 FISH—in Three Dimensions—3D-FISH ........................................ 201
Chapter 19 FISH—On Fibers .............................................................................207
Chapter 20 FISH—and Single-Cell Gel Electrophoresis Assay
(Comet Assay) .................................................................................. 211
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Contents
ix
Chapter 21 Molecular Karyotyping .................................................................... 225
Chapter 22 FISH—Mitochondrial DNA ............................................................ 251
Chapter 23 FISH—in Birds ................................................................................ 263
Chapter 24 FISH—in Fish Chromosomes .......................................................... 281
Chapter 25 FISH—and the Characterization of Synaptonemal Complex.......... 297
Chapter 26 RNA-FISH—on Lampbrush Chromosomes: Visualization of
Individual Transcription Units .........................................................307
Chapter 27 FISH—in Insect Chromosomes ....................................................... 319
Chapter 28 FISH—in Plant Chromosomes......................................................... 339
Chapter 29 FISH—and CRISPR/CAS9.............................................................. 353
Index...................................................................................................................... 357
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1
Cytogenetics and
Chromosomics
CONTENTS
Introduction................................................................................................................ 1
Chromosomics and Cytogenomics ............................................................................ 1
Application Fields of (Molecular) Cytogenetics........................................................ 2
Classical/Solid Staining .................................................................................... 3
C-Banding and NOR Silver Staining................................................................ 3
Banding Cytogenetics....................................................................................... 3
Fluorescence In Situ Hybridization (FISH)...................................................... 3
Primed In Situ Hybridization (PRINS)............................................................. 4
Comparative Genomic Hybridization (CGH)................................................... 4
Molecular Combing .......................................................................................... 4
Conclusions................................................................................................................ 4
References.................................................................................................................. 4
INTRODUCTION
This chapter is a general introduction to the book Cytogenetics and Molecular
Cytogenetics published in this new “Medical Genomics and Proteomics” book series.
The specifc topic of this book on (molecular) cytogenetics is embedded in the feld
of chromosomics, which can be only realized based on cytogenomic approaches.[1, 2]
CHROMOSOMICS AND CYTOGENOMICS
The idea of chromosomics, as an overarching designation to integrate all research
directions on the (human) genome, was introduced by Prof. Uwe Claussen (Jena,
Germany) in 2005.[3] Chromosomic research is about all genetics-related presentations of life, including DNA-basepair to chromosome- and interphase-nucleus level,
but also epigenetic aspects (including three-dimensional morphologically of nuclei,
micro-RNAs, imprinting, etc.), breakpoint characteristics and interspecies genomic
studies. Overall, chromosomics has the goal of introducing novel ideas and concepts
in biology and medicine.[1–3]
To get closer to this noble goal, cytogenomic approaches are needed. In
Table 1.1, a list of the most commonly used cytogenomic techniques is provided.[4]
Cytogenetics and molecular cytogenetics were, together with classical approaches of
1
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2
Cytogenetics and Molecular Cytogenetics
TABLE 1.1
Cytogenomic Approaches Adapted acc. to[4] Are Listed Here
Cytogenomic Field
Cytogenomic Technique
Cytogenetics
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Molecular Cytogenetics
Molecular Genetics
(classical approaches)
Molecular Genetics
(modern approaches)
Others
–
Classical/solid staining
C-banding
NOR silver staining
Banding cytogenetics
Fluorescence in situ hybridization
Primed in situ hybridization
Comparative genomic hybridization (CGH)
Molecular combing
Restriction fragment polymorphism analyses
DNA-cloning in vectors
Blotting approaches like Southern blotting
DNA-fngerprinting
PCR approaches incl. MLPA
Sanger sequencing
Array-CGH/chromosomal microarray analyses (CMA)
Second generation sequencing
Third generation sequencing
Gene editing (CRISP/CAS9)
Electron microscopy–based approaches
Laser scanning–based approaches
Optical mapping approaches
Uniparental disomy/Epigenetic changes oriented approaches—incl.
studies on long non-coding RNAs, etc.
Bioinformatics
molecular genetics, the frst possibilities of chromosomics. Groundbreaking insights
into the secrets of inheritance were and are still provided by these basic cytogenomic tools.[1, 4] Without chromosome numbers being known, including information about sex-determination systems, modern (high-throughput) molecular genetic
approaches are (if at all) only partly informative.[5, 6] It is a truism, which cannot be
repeated enough especially in the human genetics feld, that no classical cytogenomic technique is ever outdated.[7] Each approach has advantages and limitations,
which can be substituted by each other in the optimal case. Thus, to answer questions
in chromosomic research and/or diagnostics, a sensible combination of cytogenomic
approaches to be applied is always necessary.[7]
APPLICATION FIELDS OF (MOLECULAR) CYTOGENETICS
Here the techniques listed in Table 1.1 for the cytogenomic felds (i) cytogenetics and
(ii) molecular cytogenetics are treated, and it is shown in some examples where these
approaches are necessary in routine diagnostics and chromosomic research.
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Cytogenetics and Chromosomics
3
CLASSICAL/SOLID STAINING
Classical solid Giemsa or Orcein staining—also called classical cytogenetics[8]—is
the basic approach used to determine constitutional chromosome numbers in species previously not studied by cytogenetics.[9] Also, there are species in which chromosome banding is not applicable[10, 11]; here classical cytogenetics is necessary in
research.
Solid chromosome stains are also applied in research settings of radiation or
mutagenesis—and here also diagnostic applications are reported to determine number of chromosomal breaks per metaphases after irradiation and/or exposure to a
mutagen.[12]
C-BANDING AND NOR SILVER STAINING
C-banding is applied to visualize the heterochromatic regions of genomes in cytogenetic preparations, including centromeres; the latter is eponymous for this approach.
NOR silver staining highlights the location of active nucleolus organizing region(s)
in a genome.[13]
Both techniques are done in initial, research-oriented cytogenetic studies to characterize the karyotype of new plant or animal species.[9] Also they are applied in
many routine settings of cytogenetic pre- and postnatal diagnostics.[13] The latter
helps defning if an aberrant chromosome banding pattern (see next) may be just due
to a heteromorphism of heterochromatic DNA in pericentromeric, acrocentric-p-arm
or Yq12 regions of the human genome.[14]
BANDING CYTOGENETICS
Banding cytogenetics was introduced by Lore Zech in the 1970s.[15] Nowadays most
countries apply GTG-banding = G-bands by trypsin using Giemsa for routine banding cytogenetics in prenatal, postnatal and tumor cytogenetic diagnostics. Besides,
banding cytogenetics is used in animal cytogenetics, in case the corresponding chromosomes allow for introduction of a protein-based banding pattern.[16]
FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
Fluorescence in situ hybridization (FISH) is one of the major topics of this book; so
here are just some general statements. FISH is an approach that enables the in situ
localization and mapping of specifcally defned DNA-sequences.[1, 2] It is indispensable in mapping of a genome, as sequencing alone is yet insuffcient to reconstruct
a karyotype.[5] The latter is due to the fact, that highly repetitive regions of genomes
cannot be accessed by routine approaches, even though frst tools are available also
to solve that problem.[17] Still, in most cases based on NGS data, an end of chromosome cannot be distinguished from a high-repeat copy number region being typical
for a centromere. Overall, FISH is a highly fexible approach, which can be adapted
to many research and diagnostic questions, e.g. by multicolor-approaches that enable
targeting many different DNA-sequences at a time.[1, 2, 18]
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4
Cytogenetics and Molecular Cytogenetics
PRIMED IN SITU HYBRIDIZATION (PRINS)
Primed in situ hybridization (PRINS) is the second technique, besides FISH, originally included in the feld of molecular cytogenetics. As this approach exclusively
worked for repetitive sequences, it is nowadays practically no longer applied, even
though it has been used both in research and diagnostics.[19]
COMPARATIVE GENOMIC HYBRIDIZATION (CGH)
Comparative genomic hybridization (CGH) is a variant of FISH.[20] However, originally here two differently labelled while human genomes are hybridized to a normal
human metaphase plate. Thus, gains or losses in e.g. a tumor probe can be detected.
In diagnostics, this approach is nowadays replaced by array-CGH/chromosomal
microarray analyses (CMA), providing higher resolution and accuracy.[21]
In evolution research, CGH is still a great tool to, e.g. get insights into more stable
compared to regions undergoing more rapid evolution in two closely related species.[6]
MOLECULAR COMBING
So-called fber-FISH is a several decades–old variant of FISH, where DNA-probes
are hybridized instead of on interphases or metaphases on to extended DNA-fbers,
providing thus a higher in situ resolution. Recently, fber-FISH on extremely stretched
DNA-fbers became available as a standardized protocol, and is commercially available as “molecular combing”. This enables applications in research and in diagnostics for studying repetitive DNA-stretches yet not reliably accessible by any other
cytogenomic approach.[22]
CONCLUSIONS
Chromosomic research and diagnostics, in general are enabled and in parts motivated by new technical developments.[4] New cytogenomic approaches are always
welcome; however, due to specifc advantages of each technique, older ones should
not be considered too hastily as outdated.[7] As shown exemplarily here and with
no claim to completeness, cytogenetics and molecular cytogenetics are still, and
also will be in future, the most relevant participants in the concert of actual cytogenomic approaches being necessary to get as comprehensive as possible chromosomic
insights.[1, 2]
REFERENCES
1. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
2. Liehr, T. From human cytogenetics to human chromosomics. Int J Mol Sci. 2019, 20,
E826.
3. Claussen, U. Chromosomics. Cytogenet. Genome Res. 2005, 111, 101–106.
4. Liehr, T. A Defnition for cytogenomics: Also may be called chromosomics. In:
Cytogenomics; Liehr, T., Ed. Academic Press, London, 2021, pp. 1–7.
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Cytogenetics and Chromosomics
5
5. Reichwald, K.; Petzold, A.; Koch, P.; Downie, B.R.; Hartmann, N.; Pietsch, S.; Baumgart,
M.; Chalopin, D.; Felder, M.; Bens, M.; Sahm, A.; Szafranski, K.; Taudien, S.; Groth, M.;
Arisi, I.; Weise, A.; Bhatt, S.S.; Sharma, V.; Kraus, J.M.; Schmid, F.; Priebe, S.; Liehr,
T.; Görlach, M.; Than, M.E.; Hiller, M.; Kestler, H.A.; Volff, J.N.; Schartl, M.; Cellerino,
A.; Englert, C.; Platzer, M. Insights into sex chromosome evolution and aging from the
genome of a short-lived fsh. Cell. 2015, 163, 1527–1538.
6. Yano, C.F.; Sember, A.; Kretschmer, R.; Bertollo, L.A.C.; Ezaz, T.; Hatanaka, T.; Liehr,
T.; Ráb, P.; Al-Rikabi, A.; Ferreira Viana, P.; Feldberg, E.; de Oliveira, E.A.; Toma,
G.A.; Cioff, M.d.B. Against the mainstream: Exceptional evolutionary stability of l ZW
sex chromosomes across fsh families Triportheidae and Gasteropelecidae (Teleostei:
Characiformes). Chromosome Res. 2021, 29, 391–416.
7. Liehr, T.; Mrasek, K.; Klein, E.; Weise, A. Modern high throughput approaches are not
meant to replace ‘old fashioned’ but robust techniques. J. Genet. Genomes. 2017, 1, e101.
8. Liehr, T. “Classical cytogenetics” is not equal to “banding cytogenetics”. Mol Cytogenet.
2017, 10, 3.
9. Chaiyasan, P.; Mingkwan, B.; Jantarat, S.; Suwannapoom, C.; Cioff, M.d.B.; Liehr, T.;
Talumphai, S.; Tanomtong, A.; Supiwong, W. Classical and molecular cytogenetics of
Belontia hasselti (Perciformes: Osphronemidae): Insights into the ZZ/ZW sex chromosome system. Biodiversitas. 2021, 22, 546–554.
10. D’Amato, G.; Bianchi, G.; Capineri, R.; Marchi, P. N-band staining in plant chromosomes with a HCl-Giemsa technique. Caryologia. 1979, 32, 455–459.
11. Martínez-Lage, A.; González-Tizón, A.; Méndez, J. Characterization of different chromatin types in Mytilus galloprovincialis L. after C-banding, fuorochrome and restriction endonuclease treatments. Heredity. 1994, 72, 242–249.
12. Natarajan, A.T. Chromosome aberrations: Past, present and future. Mutat. Res. 2002,
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13. Weise, A.; Liehr, T. Cytogenetics. In: Cytogenomics; Liehr, T., Ed. Academic Press,
London, 2021, pp. 25–34.
14. Liehr, T. Cases with heteromorphisms. http://cs-tl.de/DB/CA/HCM/0-Start.html
[accessed on 01/12/2022].
15. Schlegelberger, B. In memoriam: Prof. Dr. rer. nat. Dr. med. h.c. Lore Zech; 24.9.1923–
13.3.2013: Honorary member of the European Society of Human Genetics, Honorary
member of the German Society of Human Genetics, Doctor laureate, the University of
Kiel, Germany. Mol. Cytogenet. 2013, 6, 20.
16. Claussen, U.; Michel, S.; Mühlig, P.; Westermann, M.; Grummt, U.W.; KromeyerHauschild, K.; Liehr, T. Demystifying chromosome preparation and the implications for
the concept of chromosome condensation during mitosis. Cytogenet. Genome Res. 2002,
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17. Nurk, S.; Koren, S.; Rhie, A.; Rautiainen, M.; Bzikadze, A.V.; Mikheenko, A.; Vollger,
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Logsdon, G.A.; Alonge, M.; Antonarakis, S.E.; Borchers, M.; Bouffard, G.G.; Brooks,
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Durbin, R.; Dvorkina, T.; Fiddes, I.T.; Formenti, G.; Fulton, R.S.; Fungtammasan, A.;
Garrison, E.; Grady, P.G.S.; Graves-Lindsay, T.-A.; Hall, I.M.; Hansen, N.F.; Hartley,
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Cytogenetics and Molecular Cytogenetics
Sullivan, B.A.; Thibaud-Nissen, F.; Torrance, J.; Wagner, J.; Walenz, P.P.; Wenger, A.;
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18. Liehr, T. Basics and literature on multicolor fuorescence in situ hybridization application. http://cs-tl.de/DB/TC/mFISH/0-Start.html [accessed on 01/12/2022].
19. Pellestor, F. Development and adaptation of the PRINS technology: An overview.
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20. Kallioniemi, A.; Kallioniemi, O.P.; Sudar, D.; Rutovitz, D.; Gray, J.W.; Waldman, F.;
Pinkel, D. Comparative genomic hybridization for molecular cytogenetic analysis of
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genetics and genome rearrangements. In: Cytogenomics; Liehr, T., Ed. Academic Press,
London, 2021, pp. 47–72.
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