A telomere is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Although there are different architectures, telomeres, in a broad sense, are a widespread genetic feature most commonly found in eukaryotes. In most, if not all species possessing them, they protect the terminal regions of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing DNA repair systems from mistaking the very ends of the DNA strand for a double strand break.
A karyotype is a preparation of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, sorted by length, centromere location and other features and for a test that detects this complement or counts the number of chromosomes. Karyotyping is the process by which a karyotype is prepared from photographs of chromosomes, in order to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities.
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
Cytogenetics is essentially a branch of genetics, but is also a part of cell biology/cytology, that is concerned with how the chromosomes relate to cell behaviour, particularly to their behaviour during mitosis and meiosis. Techniques used include karyotyping, analysis of G-banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH).
Comparative genomic hybridization(CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.
Subtelomeres are segments of DNA between telomeric caps and chromatin.
A minichromosome is a small chromatin-like structure resembling a chromosome and consisting of centromeres, telomeres and replication origins but little additional genetic material. They replicate autonomously in the cell during cellular division. Minichromosomes may be created by natural processes as chromosomal aberrations or by genetic engineering.
A dicentric chromosome is an abnormal chromosome with two centromeres. It is formed through the fusion of two chromosome segments, each with a centromere, resulting in the loss of acentric fragments and the formation of dicentric fragments. The formation of dicentric chromosomes has been attributed to genetic processes, such as Robertsonian translocation and paracentric inversion. Dicentric chromosomes have important roles in the mitotic stability of chromosomes and the formation of pseudodicentric chromosomes. Their existence has been linked to certain natural phenomena such as irradiation and have been documented to underlie certain clinical syndromes, notably Kabuki syndrome. The formation of dicentric chromosomes and their implications on centromere function are studied in certain clinical cytogenetics laboratories.
Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions you are looking for. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.
Flow-FISH is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.
Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.
Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provides comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), insert fragments into a vector to be placed as probes within a microarray, then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species.
A mega-telomere, is an extremely long telomere sequence that sits on the end of chromosomes and prevents the loss of genetic information during cell replication. Like regular telomeres, mega-telomeres are made of a repetitive sequence of DNA and associated proteins, and are located on the ends of chromosomes. However, mega-telomeres are substantially longer than regular telomeres, ranging in size from 50 kilobases to several megabases.
Spatial transcriptomics is an overarching term for a range of methods designed for assigning cell types to their locations in the histological sections. This method can also be used to determine subcellular localization of mRNA molecules. The term is a variation of Spatial Genomics, first described by Doyle, et al., in 2000 and then expanded upon by Ståhl et al. in a technique developed in 2016, which has since undergone a variety of improvements and modifications.
Physical map is a technique used in molecular biology to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter.
