Guide Advances in Enzymology and Related Areas of Molecular Biology, Volume 37

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Skip to content. Search for books, journals or webpages All Pages Books Journals. Home Books Series: The Enzymes. The treatment of cells with a hypotonic solution facilitated better chromosome spreading, leading to better definition for counting the chromosomes. Previous studies have shown that unspread and tangled chromosomes make it difficult to count the number of mammalian chromosomes in a preparation Matthey, An improved hypotonic treatment technique hypotonic shock was then applied to examine lung fibroblasts in human embryos, thereby establishing the correct modal number of 46 chromosomes in human diploid cells Tjio and Levan, In decades prior to this discovery, a human chromosome number of 48 had been described in a number of reports see Gartler, This number was based on an examination of chromosome preparations of human spermatogonia, which suggested that humans had 48 chromosomes Painter, Although only a few chromosome details were known during the pre-banding era, the chromosomes themselves could be arranged in different groups based on their sizes and centromere positions.

Following the determination of the correct modal chromosome number, the identification of the first inherited chromosomal abnormality aneuploidy leading to human diseases in man was identified. Lejeune et al. During the same period, the first acquired chromosome anomaly Philadelphia chromosome was described in patients with chronic myeloid leukemia Nowell, Subsequent technical improvements in cytogenetics included the use of phytohemagglutinin a substance that stimulates the division of T lymphocytes in vitro and the introduction of banding techniques at the end of the s.

Banding techniques use chemical treatments to produce differentially stained regions on chromosomes. The banding pattern is highly characteristic for each chromosome and facilitates the complete identification of the human karyotype. Chromosome Banding Techniques.

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With the possibility of more specific identification and detailed analyses of human chromosomes, a new phase in cytogenetics began. The first method for the visualization of a pattern of bands on human chromosomes was Q-banding Caspersson et al. Subsequently, G-banding Seabright , a technique based on the application of trypsin a proteolytic enzyme using Giemsa staining, was developed, and this method is still the most widespread cytogenetic method routinely used in clinical settings.

Classical cytogenetics became a traditional powerful diagnostic tool for detecting genomic aberrations, including both gains and losses of segments of the genome and rearrangements within and between chromosomes.

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However, the resolution of standard cytogenetics techniques remained limited, with a count of approximately bands per haploid genome Figure 1. The approaches described above facilitated the identification of structural chromosomal aberrations of at least Mb in size. The average resolution depends on different elements, such as the optical characteristics of the microscope, the complex manner in which the DNA is packaged into chromosomes and the quality of the metaphase preparations. The resolution of the standard karyotype was improved after the introduction of high-resolution banding based on the use of synchronized lymphocyte cultures Yunis, Using this technique, it was possible to increase the number of cells in the pro-metaphase or prophase stages.

Detailed principles, protocols and potential applications for these cytogenetic banding techniques have been summarized elsewhere Wegner, The first applications of molecular techniques to chromosome slide preparations, called in situ hybridization ISH , were attempts to identify and locate specific nucleic acid sequences inside cells or on chromosomes Gall and Pardue, ; John et al.

The ISH technique was based on the discovery that radioactively labeled ribosomal RNA hybridized to acrocentric chromosomes. The hybridization was visualized using autoradiography, which had been applied to human chromosomes since the early s German and Bearn, The use of ISH technology provided another dimension to the study of chromosomes, facilitating the visualization of DNA or complementary RNA sequences on chromosomes and in cells at the molecular level.

However, the use of this method was limited due to the use of radioactive isotopes, highly repetitive DNA sequences and corresponding RNA in the satellite regions of chromosomes and centromeres Pardue and Gall, Subsequently, Langer et al. The hybridization DNA probe and target sequence could be visualized through avidin or streptavidin fluorescent labeling. The development of fluorescent molecules led to direct combined with a fluorochrome or indirect through an intermediate molecule incorporated into a probe binding to DNA bases, which eventually evolved into fluorescence in situ hybridization FISH.

FISH increased the resolution at which chromosome rearrangements could be identified at submicroscopic levels, making this technique applicable for both clinical diagnosis and research. FISH has been a driving force in the further development of cytogenetic techniques.


The basic principle of FISH is that a target DNA in cells, nuclei or metaphase chromosomes is fixed and denatured on the surface of the slide. The speed of the hybridization between the probe and the target DNA varies depending on the probe used. When a non-fluorescent hapten is used e. After washing, an anti-fade solution containing DAPI 4', 6-diamidinophenylindole is applied to the slide, and a coverslip must be added.

DAPI is a fluorescent stain used extensively in fluorescence microscopy. FISH signals are typically observed using epifluorescence microscopes with specific filters for identifying fluorochromes Marcus, ; Reichman, , a charge-coupled device CCD camera captures the image and the fluorescent signals are subsequently quantified Hiraoka et al. The resulting images can be analyzed using commercially available systems. FISH provides the option for the simultaneous use of one or more DNA probes, and these probes can be distinguished after labeling with different colors or color combinations.

The probes primarily determine the resolution of these molecular cytogenetic techniques and can be classified according to the pattern of detected DNA sequences. Currently, a range of commercial probes e. FISH is a flexible technique that has driven the further development of other cytogenetic techniques. There are multiple approaches using FISH-based methods for different applications, e.

The limitation of standard FISH, however, is that it is not possible to simultaneously detect all of the chromosomes in the entire genome. The simultaneous staining of each of the 24 human chromosomes with a different color involves the use of whole-chromosome painting WCP probes, and all three of these FISH techniques use similar probe sets.

The required 24 color combinations can be achieved through combinatorial or ratio labeling. The most important aspect of these techniques is the acquisition and measurement of the complete emission spectra between and nm, rendering a unique image that contains specific spectral information for each image point.

A high-resolution molecular cytogenetic technique for the analysis of metaphase chromosomes, called multicolor banding MCB , has been proposed, which involves the microdissection of chromosomal loci to obtain a set of probes that produce multicolor pseudo-G-banding Liehr et al.

For either standard or advanced FISH methods, the preparations should be analyzed using a well maintained and calibrated fluorescence microscope equipped with the optical filter sets appropriate for the fluorochromes used and an image-recording system. The development of numerous FISH protocols and multiple approaches is the result of the efforts of many diagnostic and research scientists from different research groups worldwide. These techniques have been continuously improved, and it is not possible to cover every modification of FISH in this manuscript. Chromosomal CGH is based on quantitative two-color FISH and overcomes the problems of tissue culture failure and artifacts because this method is based on using tumor DNA extracted directly from either fresh or archival tumor tissue Kallioniemi, The major advantage of CGH over standard FISH techniques is that only the DNA from the tumor cells is needed for analysis, avoiding the difficulties of obtaining metaphase chromosomes with good morphology and resolution for the analysis.

In CGH, total genomic DNA obtained from control cells and test samples is differentially labeled using green fluorescein isothiocyanate, FITC and red Texas red fluorescent dyes, denatured, co-precipitated in the presence of blocking DNA to suppress repetitive sequences and subsequently co-hybridized to normal metaphase chromosomes.

Due to the simultaneous hybridization to normal denatured metaphase chromosome spreads, there is competition for DNA hybridization to homologous sites. After hybridization and washing, the metaphase spreads are observed under a fluorescent microscope, and image analysis is performed using image analysis software. The resulting fluorescence intensities of the test and reference hybridizations are digitally quantified along the length of each chromosome.

Chromosomal regions equally represented in both the test and reference samples appear yellow because of the presence of an identical amount of red and green dye, while regions with copy number loss are red and have a ratio below one Figure 3a. More recently, the development of array-based CGH array-CGH approaches involving the substitution of metaphase chromosomes with DNA sequences adhered onto glass slides has increased the resolution for detecting copy number changes in the human genome, leading to more detailed information on genomic gains and losses Figure 3b.

Among all of the recent advances in techniques for examining chromosomes, array-CGH technology has been suggested as a technique that will gradually replace classical cytogenetics in clinical diagnosis. Indeed, the process involves comparative genomic hybridization using an array rather than a metaphase spread as the substrate Solinas-Toldo et al. The actual microarray comprises thousands of spots of reference DNA sequences applied in a precisely gridded manner on the slide. As the resolution of the array yields improves, shorter sequences have been used as targets, including smaller cDNA fragments Pollack et al.

Furthermore, ar-ray-CGH provides resolution at the nucleotide level. Single-nucleotide polymorphism arrays SNP arrays have the highest resolution kb of all of the available array-based platforms see Le Scouarnec and Gribble, In addition to CNVs, the genotype information obtained from SNP arrays enables the detection of stretches of homozygosity and thus the identification of recessive disease genes, mosaic aneuploidy or uniparental disomy UPD de Leeuw et al. While only SNP arrays enable the detection of copy number-neutral regions in the absence of heterozygosity AOH , these arrays have limited ability to detect single-exon copy CNVs due to the distribution of SNPs across the genome.

Combining both array-CGH and SNP genotyping in a single platform optimizes the clinical diagnostic capability, offering the simultaneous detection of copy number neutral and small intragenic copy number changes Wiszniewska et al. The number, size and distribution of the DNA segments on the glass slide determine the array resolution, but commonly, the higher the number of DNA fragments, the higher the resolution.

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According to Balliff et al. Currently, there are several different commercially available diagnostic DNA microarray platforms comparing thousands of DNA sequences from a patient sample with reference control DNA samples or control datasets to detect chromosomal CNVs. Recently, a modified array protocol, called translocation CGH tCGH , was developed to address recurrent translocation breakpoints in hematological neoplasms.

Prior to the hybridization step in the array procedure, a linear PCR amplification is performed across the known recurrent translocation breakpoints in hematological neoplasms. Thus, it is possible to detect copy number changes and known recurrent translocations near or at the breakpoints Greisman et al.

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Custom-made commercial arrays that use general standard protocols can also be ordered. Detailed information on the protocols and references is available elsewhere Banerjee and Shah, Several decades ago, molecular methods were introduced into cytogenetic studies, facilitating the development of new applications, many of which were used diagnostically or as prognostic tools in medicine. Furthermore, molecular cytogenetic approaches have also become indispensable for a range of research purposes.

The use of molecular techniques in cytogenetic studies is increasing, and the many variations, adaptations and specifications make it challenging to cover all of the possible applications. Since the introduction of FISH in the late s, there has been a tremendous increase in the number of studies using molecular approaches in cytogenetics to detect chromosomal abnormalities and evaluate CNVs in the human genome. FISH offers numerous possibilities for studying either the whole genome or specific genomic loci regions , and this technique has been widely used to detect aneuploidies and recurrent chromosomal abnormalities in preimplantation genetic, prenatal, and postnatal diagnoses and cancer cytogenetics.

Moreover, the application of FISH has long been demonstrated as extremely valuable for studying chromosomal and genome organization, evolution and variations in health and disease see Geurts and de Jong ; McNamara et al. A significant advantage of FISH is that it can be applied in non-dividing cells, thereby facilitating the direct investigation of chromosomes in cytological preparations and tissue sections. Classical cytogenetic analysis depends on cells undergoing mitosis to obtain metaphase chromosome spreads. Therefore, cells must be cultured in vitro either as a short-or long-term culture.

Thus, interphase FISH on uncultured amnion cells has become a useful method for the rapid and early diagnosis of the most common chromosome disorders trisomies 21, 13, 18 and sex chromosome aneuploidies in fetal cells Eiben et al. For prenatal aneuploidy screening using uncultured amniocytes, no time-consuming cell culture is required, and the results can be obtained within hours. Three satellite centromeric probes for chromosomes X, Y and 18 and two locusspecific probes for the 13q14 and 21q Interphase FISH in prenatal diagnosis is a quick, accurate, sensitive and relatively specific method to detect aneuploidies in samples of uncultured chorionic villus Rosner et al.

In addition, using locus-specific probes, FISH has also been used to confirm clinical diagnoses of known microdeletion and microduplication syndromes Riegel and coworkers, unpublished data. However, FISH has limitations in the detection of known microdeletion syndromes. Occasionally, patients with small and unusual deletions might escape detection, depending on the specificity of the fluorescent probe.

Moreover, cases with gene or imprinting mutations, occurring in some microdeletion syndromes, e. The analysis of telomeres using FISH techniques has been conducted in cancer and aging research telomere biology ; however, due to the lack of specificity of the DNA probes TTAGGG repetitive sequence motifs , this technique is poorly applicable for diagnosis Aubert and Lansdorp, Multicolor FISH approaches have been most valuable for cancer cytogenetics, but these methods have also been applied to diagnose constitutional chromosomal abnormalities Liehr et al.

Applications of CGH Analysis. Although CGH has primarily been applied to study solid tumors, this technique has also used to study leukemia and lymphoma Kallioniemi et al. However, given that CNVs are associated with many conditions, ranging from cancer to developmental abnormalities, CGH has also been applied to identify constitutional chromosomal abnormalities in clinical samples Daniely et al. Several reports have demonstrated the use of either standard CGH or array-CGH to detect chromosomal abnormalities in single cells of pre-implantation embryos Wells and Delhanty, ; Le Caignec et al.

Array-CGH was initially applied to identify chromosomal imbalances through the detection of CNVs in tumors to distinguish candidate genes involved in the pathogenesis of cancer Cai et al. In clinical diagnostics, both oligonucleotide array-CGH and SNP genotyping have been demonstrated as powerful genomic technologies for evaluating idiopathic mental retardation MR also referred to as developmental delay DD , intellectual disability ID or learning difficulty , associated congenital abnormalities MCA , autistic spectrum disorders ASDs , schizophrenia and other neuropsychiatric disorders.

Furthermore, the introduction of genomewide array platforms facilitated the detection of chromosomal abnormalities consistent with genetic syndromes at earlier ages, when only a few clinical findings might be present. In , two studies employing array-based platforms revealed that CNVs exist in many large DNA genomic segments between normal human individuals, suggesting that these variations are fairly common and might represent polymorphic variations and a significant source of genetic variation Iafrate et al.

Furthermore, the examination of the genomic content of CNVs revealed that these genomic regions include many functional genes involved in the regulation of cell growth and metabolism Iafrate, , implicating CNVs in human traits, disease and evolution. Since that time, many additional studies using a multitude of different high-resolution genomeanalysis platforms have advanced our knowledge regarding CNVs.

Since Vissers et al. The rate differences might reflect differences in the resolutions of the array platforms used, the criteria for patient selection and the interpretation of the clinical relevance of the CNVs detected. Letter 23 September In addition to causing A-to-G base transitions, adenine base editors also cause C base substitutions. Letter 09 September Preservation of human livers at subzero temperatures with ice-free supercooling extends ex vivo organ life. Dereplication by inactivation of antibiotic production clusters in actinomycetes enables antibiotic discovery.

Correspondence 19 September Respiratory pathogens and the human immune response they elicit can now be studied in humanized mice. Livers are stored in a supercooled state for extended periods, an approach that could expand access to liver transplantation. News 09 September News in Brief 04 September Duke University.

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