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Examples of different types of fluorescence in situ hybridisation (FISH) probes. (a) Genespecific probes target specific nucleic acid sequences on a chromosome. (b) Centromeric probes bind to repetitive sequences that are specific to the centromeric regions. (c) Telomeric probes recognise the repetitive sequence TTAGGG, and can be used to visualise all telomeres simultaneously. Chromosome-specific telomeric probes hybridise to subtelomeric, chromosome-specific repeats. (d) Chromosome-painting probes consist of pools of chromosome-specific probes (fig001trn).
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Molecular cytogenetic techniques that are based on fluorescence in situ hybridisation (FISH) have become invaluable tools for the diagnosis and identification of the numerous chromosomal aberrations that are associated with neoplastic disease, including both haematological malignancies and solid tumours. FISH can be used to identify chromosomal rea...
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... because the size of the DNA region that has been deleted is often too small to be detected by conventional banding techniques. FISH probes are highly specific for their target or cDNA sequence, and can be divided into four main types: gene-specific probes, repetitive- sequence probes, whole-genomic DNA probes and chromosome-painting probes (see Fig. ...
Similar publications
In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and compar...
Clinical and cancer cytogenetics is a rapidly evolving discipline. The past decade has seen a dramatic change in molecular biology and fluorescence microscopy. The use of fluorescence in situ hybridization (FISH) technologies has enabled the rapid analysis of cytogenetic specimens as an adjunct to classical cytogenetic analysis. Spectral karyotypin...
The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping t...
Citations
... The whole chromosome painting probes or arm-specific sequence probes use mixtures of fluorescently labeled DNA sequences derived from the entire length of the specific chromosome or one of its arms. [16] These probes can only be applied to metaphase cells because in interphase cells, they generate large and diffuse signals. They are helpful in characterizing complex rearrangements and marker chromosomes. ...
... Gene-specific or locus-specific probes are derived from unique DNA sequences or loci within the chromosome. Even using banding techniques on highly stretched (prometa-phase) chromosomes, the smallest detectable chromosome abnormality is 2000-3000 kb, whereas gene-or locus-specific probes can consistently detect segments as small as 0.1 Mb. [16] Thus, these probes are widely used in both clinical and basic research. Locus-specific probes have been useful in gene mapping and characterizing structural rearrangements, amplifications, and origin of marker chromosomes. ...
During the last five decades, chromosome analysis identified recurring translocations and inversions in leukemias and lymphomas, which led to cloning of genes at the breakpoints that contribute to oncogenesis. Such molecular cytogenetic methods as fluorescence in situ hybridization (FISH), copy number (CN) arrays or optical genome mapping (OGM) have augmented standard chromosome analysis. The use of both cytogenetic and molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR) and next generation sequencing (NGS), including whole-genome sequencing (WGS), discloses alterations that not only delineate separate WHO disease entities but also constitute independent prognostic factors, whose use in the clinic improves management of patients with hematologic neoplasms.
... trisomy 19) in this metaphase distribution, derived from a glial cell taken from a mouse's brain, chromosome painting probes were used. Chromosome 19 is green in colour(McNeil and Ried 2000). ...
This paper aimed to understand and compare the two popular cytogenetic techniques of fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in detecting breast cancer chromosomal abnormality. Several chromosomal anomalies play a role in the development of breast cancer, and the two above approaches play an important role in confirming fluorescence in situ hybridization in particular (FISH). However, comparative genomic hybridization has developed DNA copy number profiles for most of the publicly available breast cancer cell lines for the FISH methods rely on the fluorescent probes. Chromosomal profiles can be generated for the suspected chromosomal abnormality, copy number changes between the tumour and the DNA control can be compared, and the results can be registered. Today, modern cytogenetic tools such as fluorescence in situ hybridization (FISH) are more commonly used to detect any microdeletion that cannot be detected by conventional cytogenetic karyotypes that involve a high rate of cell division and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and research. Usually, this is a problem for physicians, and there are still many drawbacks and disadvantages concerning the high benefits, such as false findings. Normal chromosome in situ hybridization requires the hybridization of a labelled DNA probe into denatured chromosomal DNA present in metaphase chromosomes in an air-dried microscope slide preparation. Metaphase spreads are used for traditional chromosome FISH (metaphase FISH). Positive and positive signs of hybridization also appear as a double spot, corresponding to the hybridized probe for both sister chromatids. A further extension of chromosome painting is comparative genomic hybridization (CCI-I). CCH involves simultaneous chromosome painting in two different colours using complete DNA from two similar sources as probes, which reveal variations concerning the benefit or loss of sub-chromosomal regions or even entire chromosomes.
... This technique involves the use of DNA or RNA probes, which are labelled with fluorescent molecules and hybridized to genomic DNA sequences, to enable the study of specific sites on chromosomes. It can be used in physical chromosome mapping, chromosomes rearrangement analysis, comparative gene mapping, studies of chromosome structure and evolution and a host of other interesting areas [31,46,47,48,31,[46][47][48]. The in-situ methods involve the use of DNA or RNA probes, which are labelled with fluorescent molecules and hybridized to genomic DNA sequences, to enable the study of specific sites on chromosomes. ...
... This technique involves the use of DNA or RNA probes, which are labelled with fluorescent molecules and hybridized to genomic DNA sequences, to enable the study of specific sites on chromosomes. It can be used in physical chromosome mapping, chromosomes rearrangement analysis, comparative gene mapping, studies of chromosome structure and evolution and a host of other interesting areas [31,46,47,48,31,[46][47][48]. The in-situ methods involve the use of DNA or RNA probes, which are labelled with fluorescent molecules and hybridized to genomic DNA sequences, to enable the study of specific sites on chromosomes. ...
Cytogenetics is the study of chromosomes; their structure and properties, chromosome behavior during cell division, their influence on traits and factors which cause changes in chromosomes. Veterinary cytogenetics is the application of cytogenetics to clinical problems that occur in animal production. It has been applied to understand problems such as infertility and its types, embryonic and fetal death, abnormality in sexual and somatic development and hybrid sterility and also prenatal sex determination and other forms of chromosomal abnormalities. These are achieved through conventional and banded karyotyping techniques and molecular cytogenetic techniques. Although conventional techniques are still useful and very widely applied, the nature of cytogenetics has gradually changed as a result of advances achieved in the molecular cytogenetic techniques for Review Article Yahaya et al.; ARRB, 33(1): 1-16, 2019; Article no.ARRB.51281 2 example fluorescent in situ hybridization and array-based techniques. These changes are evident in both molecular diagnostics and basic research. The combination of conventional and molecular cytogenetics has given rise to high resolution techniques which have enabled the study of fundamental questions regarding biological processes. It enables the study of inherited syndromes, the mechanisms of tumorigenesis at molecular level, genome organization and the determination of chromosome homologies between species. It allows the ease with which animals are selected in breeding programs and other important aspects of animal production. In this paper we discussed a number of techniques employed in cytogenetics and their methodologies, and recommend where future focus should be for the benefits of animal production.
... Multiple methods are available to assess chromosomal stability of cells including Giemsa (G) banding, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY) and comparative genomic hybridization (CGH). SKY is a rapid FISH-based method in which chromosome-specific fluorescent labels are used to visualize all chromosomes in a single hybridization [24,25]. Using SKY, we identified genomic aberrations in bone marrow-derived hMSCs from multiple donors and at multiple passages to analyze the chromosomal stability of each cell line during culture. ...
... It readily identifies aberrant chromosomes even if their structural abnormalities or small size render them hard to recognize by G-banding [39]. SKY readily identifies translocations and complex chromosomal rearrangements between chromosomes [24,25,38]. However, it cannot evaluate certain structural abnormalities, such as inversions, deletions and duplications within the same chromosome. ...
Background aims:
Mesenchymal stromal cells (MSCs) are being investigated for use in cell therapy. The extensive in vitro expansion necessary to obtain sufficient cells for clinical use increases the risk that genetically abnormal cells will arise and be propagated during cell culture. Genetic abnormalities may lead to transformation and poor performance in clinical use, and are a critical safety concern for cell therapies using MSCs.
Methods:
We used spectral karyotyping (SKY) to investigate the genetic stability of human MSCs from ten donors during passaging.
Results:
Our data indicate that chromosomal abnormalities exist in MSCs at early passages and can be clonally propagated. The karyotypic abnormalities observed during our study diminished during passage.
Conclusions:
Karyotyping of MSCs reveals characteristics which may be valuable in deciding the suitability of cells for further use. Karyotypic analysis is useful for monitoring the genetic stability of MSCs during expansion.
... As disease progresses, the tumor cells can acquire further mutations, proliferate or commit apoptosis, thus changing the population-wide genomic pro fi le of a tumor and its cancer-hallmark. Analysis of chromosomal changes by fl uorescent in situ hybridization (FISH)-based cytogenetic approaches including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and multiplex-FISH (M-FISH) have mapped various chromosomal regions involved in various cancers [12][13][14][15][16] . Recently, microarray techniques (e.g., array-CGH or matrix-CGH), and next-generation sequencing technologies (e.g., Illumina, 454, ABI-Solid) have become widely available, hastening the speed and improving the resolution at which an oncologist can map regions of DNA sequence from the cancer tissue that are ampli fi ed or reduced compared to normal tissue. ...
BioSensors: Plasmonics
A major goal in biosensor development is the creation and commercialization of a
new, molecular, high-data-content diagnostic platform for the detection of biomarkers
for individual patient care and personalized disease treatment. The fi eld
of plasmonics offers the potential for creating such biosensors. Plasmonic substrate
design for speci fi c biomarker applications, relies on nanoscale- precise substrate
fabrication creating biosensor devices for the ultra-sensitive detection of target
analytes in biological media, e.g., blood, plasma, and other bodily fl uids.
The fi eld of plasmonics captures the physics of the interaction of light with plasmonic
oscillations of electrons in materials. Raman spectroscopy is a technique
based on the inelastic Raman scattering of incident light to give unique frequency
shifted fi ngerprints for molecules and information on their vibrational, rotational,
and electronic energies. Raman spectroscopy can take advantage of plasmonic structures
and often works over the visible or near IR range of incident light. While standard
Raman spectroscopy has a relatively low occurrence of Raman scattering
events, substrates can be created that enhance the occurrence of the Raman events
many orders of magnitude and result in high levels of sensitivity, down to the single
molecule detection regime. Surfaces, and nanosize features therein, play a main role
in what is known as Surface Enhanced Raman Spectroscopy (SERS) [ 36, 37 ]. In
addition to surface features such as surface roughness, it has been shown that several
nano- and microengineered architectures have similar or signi fi cantly greater Raman
enhancement ability. As these substrates utilize plasmonic mechanisms to achieve
extreme enhancement abilities, they are often referred to as Plasmonic Enhanced
Raman Spectroscopy (PERS) substrates. Examples include plasmonic architectures
that have nanometer scale gaps between materials that support plasmons (generally
silver and gold) [ 38 ] , micron or submicron periodicity of features, nanoparticle
systems [ 39 ], nanorings [ 40 ], nanocrescents [ 41 ], nanorods [ 42 ], and many others
... The images are analyzed by a combination of epifluorescence microscopy, charge-coupled device (CCD) imaging, and Fourier spectroscopy, which enables the analysis of all emission spectrum with a single exposure of every image points. 23,25,26 Gene screening tests These genetic tests are based on analysis of the individual gene sequence. Unlike cytogenetic tests, they are not directed at the chromosome morphology, but rather at nucleotide sequence and organization. ...
Accurate molecular diagnosis of genetic eye diseases has proven to be of great importance because of the prognostic and therapeutic value of an accurate ascertainment of the underlying genetic mutation. Efforts continue in diagnostic laboratories to develop strategies that allow the discovery of responsible gene/mutations in the individual patient using the least number of assays and economizing on the expenses and time involved in the process. Once the ophthalmologist has made the best possible clinical diagnosis, blood samples are obtained for genetic testing. In this paper we will review the basic laboratory methods utilized to identify the chromosomal or mutational etiology of genetic diseases that affect the eye.
... Initially, the probes for in situ hybridization were labeled directly by radioactive isotopes 32 P, 125 I, 3 H and 35 S, but since the beginning of the 1980 the probes started being labeled by non-radioactive molecules. Although several methods based on enzymatic reaction using alkaline phosphatase, beta-galactosidade or horseradish peroxidade were available, the most applied method in the subsequent years was based in the utilization of fluorescent elements, therefore the technique was named fluorescent in situ hybridization (FISH) , Forster et al. 1985, Pinkel et al. 1986, McNeil and Ried 2000, Schwarzacher and Heslop-Harrison 2000. The use of FISH permits a color era for cytogenetics and a substantial increase in the quality of the final results observed. ...
Decades before the recent advances in molecular biology and the knowledge of the complete nucleotide sequence of several genomes, cytogenetic analysis provided the first information concerning the genome organization. The exploration of molecular biology techniques in the cytogenetic area represents a powerful tool for advancement in the construction of physical chromosome maps of the genomes. The most important advances in cytogenetics comes from the physical anchorage of genetic linkage maps in the chromosomes through the hybridization of DNA markers onto chromosomes. This new book presents and discusses current research in the study of animal genomes under the focus of cytogenetics.
... LNA (locked-nucleic acid) or PNA probes, for more details see [3,13,37,38]), probes for molecular cytogenetic assays can be classified according to the pattern of detected DNA sequences. Such classification includes repetitive-sequence DNA (centromeric and telomeric), site-specific, whole chromosome painting (wcp) probes [3,55]. FISH, which paints repetitive genomic sequences, can be performed with either centromeric (chromosome enumeration or chromosome-specific) or telomeric DNA probes. ...
... I-FISH analysis using telomeric probes was only described in few nuclear organization studies [57]. Contrariwise, applications of I-FISH with centromeric DNA probes are an integral part of diagnostics in medical genetics, oncology and reproductive medicine1235,7,10,12,13,20212223242526272829303536373841,42,44,46,55,58596061. Moreover, application of these probes has been long demonstrated to be extremely valuable for research in fields of chromosome biology studying genome organization (chromosomal and nuclear), evolution, behavior and variation in health and disease [2,3,7,10,12, 13,202122232425262728293035,41,42,44,55,626364656667. ...
... Contrariwise, applications of I-FISH with centromeric DNA probes are an integral part of diagnostics in medical genetics, oncology and reproductive medicine1235,7,10,12,13,20212223242526272829303536373841,42,44,46,55,58596061. Moreover, application of these probes has been long demonstrated to be extremely valuable for research in fields of chromosome biology studying genome organization (chromosomal and nuclear), evolution, behavior and variation in health and disease [2,3,7,10,12, 13,202122232425262728293035,41,42,44,55,626364656667. The popularity of these DNA probes is usually attributed to near 100% hybridization efficiency because of painting highly repetitive DNAs as well as to chromosome-specifity of centromeric human DNAs allowing analysis of individual homologous chromosome pairs in interphase [7,10,30,35]. ...
Human karyotype is usually studied by classical cytogenetic (banding) techniques. To perform it, one has to obtain metaphase chromosomes of mitotic cells. This leads to the impossibility of analyzing all the cell types, to moderate cell scoring, and to the extrapolation of cytogenetic data retrieved from a couple of tens of mitotic cells to the whole organism, suggesting that all the remaining cells possess these genomes. However, this is far from being the case inasmuch as chromosome abnormalities can occur in any cell along ontogeny. Since somatic cells of eukaryotes are more likely to be in interphase, the solution of the problem concerning studying postmitotic cells and larger cell populations is interphase cytogenetics, which has become more or less applicable for specific biomedical tasks due to achievements in molecular cytogenetics (i.e. developments of fluorescence in situ hybridization -- FISH, and multicolor banding -- MCB). Numerous interphase molecular cytogenetic approaches are restricted to studying specific genomic loci (regions) being, however, useful for identification of chromosome abnormalities (aneuploidy, polyploidy, deletions, inversions, duplications, translocations). Moreover, these techniques are the unique possibility to establish biological role and patterns of nuclear genome organization at suprachromosomal level in a given cell. Here, it is to note that this issue is incompletely worked out due to technical limitations. Nonetheless, a number of state-of-the-art molecular cytogenetic techniques (i.e multicolor interphase FISH or interpahase chromosome-specific MCB) allow visualization of interphase chromosomes in their integrity at molecular resolutions. Thus, regardless numerous difficulties encountered during studying human interphase chromosomes, molecular cytogenetics does provide for high-resolution single-cell analysis of genome organization, structure and behavior at all stages of cell cycle.
... Initially, the probes for in situ hybridization were labeled directly by radioactive isotopes 32 P, 125 I, 3 H and 35 S, but since the beginning of the 1980 decade the probes started being labeled by non-radioactive molecules. Although several methods based on enzymatic reaction using alkaline phosphatase, beta-galactosidade or horseradish peroxidade were available, the most applied method in the subsequent years was based in the utilization of fluorescent elements, therefore the technique was named fluorescent in situ hybridization (FISH) (Pardue and Gall 1969, Forster et al. 1985, Pinkel et al. 1986, McNeil and Ried 2000, Schwarzacher and Heslop-Harrison 2000. The use of FISH permit a color era for cytogenetics and a substantial increase in the quality of the final results observed. ...
Decades before the recent advances in molecular biology and the knowledge of the complete nucleotide sequence of several genomes, cytogenetic analysis provided the first information concerning the genome organization. Since the beginning of cytogenetics, great effort has been applied for understanding the chromosome evolution in a wide range of taxonomic groups. The exploration of molecular biology techniques in the cytogenetic area represents a powerful tool for advancement in the construction of physical chromosome maps of the genomes. The most important contribution of cytogenetics is related to the physical anchorage of genetic linkage maps in the chromosomes through the hybridization of DNA markers onto chromosomes. Several technologies, such as polymerase chain reaction (PCR), enzymatic restriction, flow sorting, chromosome microdissection and BAC library construction, associated with distinct labeling methods and fluorescent detection systems have allowed for the generation of a range of useful DNA probes applied in chromosome physical mapping. Concerning the probes used for molecular cytogenetics, the repetitive DNA is amongst the most explored nucleotide sequences. The recent development of bacterial artificial chromosomes (BACs) as vectors for carrying large genome fragments has allowed for the utilization of BACs as probes for the purpose of chromosome mapping. BACs have narrowed the gap between cytogenetic and molecular genetics and have become important tools for visualizing the organization of genomes and chromosome mapping. Furthermore, the use of chromosome probes has permitted the development of chromosome painting technologies, allowing an understanding of particular chromosomal areas, whole chromosomes or even whole karyotypes. Moreover, chromosomal analysis using these specific probes has contributed to the knowledge of supernumerary chromosomes, sex chromosomes, species evolution, and the identification of chromosomal rearrangements. Finally, the synergy between chromosomal and molecular biology analysis makes cytogenetics a powerful area in the integration of knowledge in genetics, genomics, taxonomy and evolution.
... 18q11.2 and 20q13.1-q13.3. [5][6][7] Both methods are complementary in characterizing chromosomal aberrations but they are limited to the detection within a range of 1-20 Mb. 8 Another disadvantage is that LOH can be detected only indirectly if it is caused by deletion of one allele. The single nucleotide polymorphism (SNP) array technology, which has originally been developed for allelotyping and linkage analyses, allows a genome-wide fine mapping of copy-number changes within a range of 30-900 kb. ...
We performed genome-wide analysis of copy-number changes and loss of heterozygosity (LOH) in Barrett's esophageal adenocarcinoma by single nucleotide polymorphism (SNP) microarrays to identify associated genomic alterations. DNA from 27 esophageal adenocarcinomas and 14 matching normal tissues was subjected to SNP microarrays. The data were analyzed using dChipSNP software. Copy-number changes occurring in at least 25% of the cases and LOH occurring in at least 19% were regarded as relevant changes. As a validation, fluorescence in situ hybridization (FISH) of 8q24.21 (CMYC) and 8p23.1 (SOX7) was performed. Previously described genomic alterations in esophageal adenocarcinomas could be confirmed by SNP microarrays, such as amplification on 8q (CMYC, confirmed by FISH) and 20q13 or deletion/LOH on 3p (FHIT) and 9p (CDKN2A). Moreover, frequent gains were detected on 2p23.3, 7q11.22, 13q31.1, 14q32.31, 17q23.2 and 20q13.2 harboring several novel candidate genes. The highest copy numbers were seen on 8p23.1, the location of SOX7, which could be demonstrated to be involved in amplification by FISH. A nuclear overexpression of the transcription factor SOX7 could be detected by immunohistochemistry in two amplified tumors. Copy-number losses were seen on 18q21.32 and 20p11.21, harboring interesting candidate genes, such as CDH20 and CST4. Finally, a novel LOH region could be identified on 6p in at least 19% of the cases. In conclusion, SNP microarrays are a valuable tool to detect DNA copy-number changes and LOH at a high resolution. Using this technique, we identified several novel genes and DNA regions associated with esophageal adenocarcinoma.
