page_title: Mega-telomere
section_title: Identification Methods
hierarchical_section_title: Mega-telomere / Identification Methods
caption_attribution_description: English: Slot blot method of analysis can be utilized to determine the total amount of telomeric sequence per genome (inclusive of interstitial and terminal arrays)
context_page_description: 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.
  Telomeres act like protective caps for the chromosome. During cell division, a cell will make copies of its DNA. The enzymes in the cell that are responsible for copying the DNA cannot copy the very ends of the chromosomes. This is sometimes called the "end replication problem". If a cell did not contain telomeres, genetic information from the DNA on the ends of chromosomes would be lost with each division. However, because chromosomes have telomeres or mega telomeres on their ends, repetitive non-essential sequences of DNA are lost instead. While the chromosomes in most eukaryotic organisms are capped with telomeres, mega-telomeres are only found in a few species, such as mice and some birds.
context_section_description: A variety of cytogenetic and molecular methods have been utilized to identify and study mega-telomeres in vertebrate species. Many of these techniques allow researchers to both discover the presence of a mega-telomere in a genome but also to characterize telomere arrays.
  Cytogenetic studies employ fluorescence in situ hybridization (FISH) with telomeric probes to label telomeres on chemically-treated cells fixed to glass slides. More specifically, telomere-peptide nucleic acid fluorescein probes are frequently used to identify telomeric sequence repeats on mitotic metaphase and interphase or meiotic pachytene-stage chromosomes. FISH images allow both the identification of mega-telomeric chromosomes and the visualization of chromosome structure, GC-rich DNA regions, and, depending on the experiment, co-localization with genetic regions or genes.
  Molecular techniques for quantifying telomeric sequences include pulse-field gel electrophoresis (PFGE), slot blot, horizontal gel electrophoresis, and Contour-clamped homogeneous electric field pulse field gel electrophoresis (CHEF-PFGE). In these techniques, purified genomic DNA is isolated and digested with restriction enzymes, such as HaeIII, HinfI, AluI, Sau3AI, EcoRI, EcoRV, PstI, SstI, BamHI, HindIII or BglII, and quantified by fluorometry.
  The digestion of DNA into smaller fragments by restriction enzymes, separation of variable-sized DNA fragments via electrophoresis, and labeling of fragments containing telomeric DNA using a specific radio- or fluorescently-labeled probe are the essential steps completed within many molecular techniques. In many cases, the DNA fragments are transferred to distinctive membranes before labeling via blotting techniques (i.e. Southern blot). Specialized protocols have demonstrated the ability to isolate high molecular weight Class III telomeric DNA from Class I and II fragments as well as characterize the size ranges found within each class. The pattern of the telomeric fragments on the stained or labeled membrane is typically unique to the DNA sample (i.e. telomere arrays are rarely identical). Molecular weight markers are usually separated via electrophoresis through agarose gel along with genomic DNA fragments to aid in sizing telomeric arrays and identifying array inter- and intra- individual variability. Slot blot, however, is conducted without DNA fragmentation or separation, rather whole genomic DNA is used to quantify the total concentration of telomeric DNA. The flaw of this technique is that the size of the labelled DNA molecules cannot be identified. In slot blot (or dot blot), total genomic DNA is attached to a membrane and labeled with a telomere-probe that produces a sample-specific chemiluminescence signal, which is captured and quantified by fluorometer equipment and software. A known concentration standard must be labeled and quantified simultaneously in order to accurately determine the telomeric sequence concentration in the DNA samples.
caption: Slot blot method of analysis can be utilized to determine the total amount of telomeric sequence per genome (inclusive of interstitial and terminal arrays)
exif: {"Image XResolution": "150", "Image YResolution": "150", "Image ResolutionUnit": "Pixels/Inch", "Image ExifOffset": "78", "EXIF ExifImageWidth": "1056", "EXIF ExifImageLength": "677"}