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File:FISH (Fluorescent In Situ Hybridization) jpg - Wikipedia

File:FISH (Fluorescent In Situ Hybridization) jpg - Wikipedia



Home › Aging › Q-FISHing to monitor molecular aging By Klaus D. Linse on January 31, 2013 • ( 0 ) By Klaus D. LinseFluorescence in situ hybridization (FISH) is  a powerful tool and probes that exhibit high DNA and RNA affinities promise to  improve the sensitivity of the technique. Artificial nucleotides such as bridged nucleic acids (BNAs) have been described for the development of chimeric BNA/DNA oligonucleotides as probes for fluorescence in situ hybridization on metaphase chromosomes and interphase nuclei as well as others.Do you want to know your biological age at the molecular level?As humans, we all age and finally die. Since many of us now may live almost a century or even longer, some scientists now think that some humans could live up to 120 years. It is well known that mammals and other species have different lifespans.  Research to find out what determines life expectancy in different species but specifically in humans is an ongoing quest.A few years ago a Spanish group of scientists developed a high-throughput telomere length quantification method they called Q-FISH (Canela et al., 2007).  When this group of researchers studied the length of telomeres in cancer and age-related diseases in human populations, they found that the major limitations for their studies was the lack of suitable high-throughput assays that allowed them to measure telomere length on a lot of samples. Therefore, they set out to develop an automated high-throughput assay to measure telomere length. This assay is now referred to as the HT Q-FISH method (High-Throughput Quantitative Fluorescence In situ Hybridization).Now why would the researchers need this type of assay?The answer lies in the observation that telomers, which are special structures at the ends of eukaryotic chromosomes, protect them from degradation and DNA repair activities. The enzyme telomerase is a reverse transcriptase that elongates telomeres in the cells it is expressed in. Telomerase was found to be active in germ cells and stem cell populations but not in adult tissue cells. In normal adult tissue cells or somatic cells the telomere activity levels are not sufficient to prevent telomere shortening associated with cell division. During the lifetime of a human the number of times a normal human cell population will divide until cell division stops appear to be finite. However, cancer cell lines can almost live on forever and are considered to be immortal.The Hayflick limit and immortalityEmpirical data showed that the telomeres associated with the DNA in a cell will get slightly shorter with each new cell division until they have shortened to a critical length. In 1961 Hayflick and Moorhead discovered when studying cultured human cell lines that the cells in a cell culture will divide between 40 and 60 times. Once this number of divisions, the “Hayflick limit”, is reached the population will enter a senescence phase. Researchers now believe that telomere shortening in humans eventually makes cell division impossible. The aging of the cell population appears to correlate with the overall physical aging of the human body. Natural maintenance of the length of the telomeric region is now thought to prevent genomic instability and the development of cell mutations that may lead to cancer or other diseases. The “Telomere Shortening and Aging” theory was born. Cancer cells are said to become immortal by acquiring mutations that can occur in somatic cells over time. Whereas, immortalized cell lines are populations of cells originating from multi-cellular organisms that normally do not proliferate indefinitely. These cells have evaded normal cellular senescence and can keep continue to divide. The cells can be grown for prolonged periods of time in vitro. The mutations required for immortality can occur naturally or be intentionally induced for experimental purposes. Immortal cell lines have become important research tools.The Nobel Prize in Physiology or Medicine in 2009 was awarded to telomere researchers!In the year 2009 the Nobel Prize in Physiology or Medicine was awarded to Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak for their work in which these scientists solved a major problem in biology. The question was how the chromosomes can be copied in a complete way during cell divisions and how they are protected against degradation. The Nobel Laureates showed that the solution is to be found in the ends of the chromosomes – the telomeres – and in an enzyme that forms them – the telomerase.Mechanisms that regulate the number of cells, as well as how many times a cell can divide, have remained largely elusive but the general observation for mortal human cell lines now suggests that most human cells can replicate about 50 times before the telomeres are getting too short. This has led to the notion that telomeres are the “secret to longevity”. Furthermore, circumstances in which the telomeres will not shorten have been observed, for example, in cancer cells. Cancer cells can become immortal by switching on their telomerase, which adds to the telomeres when cells divide. Some cells in a human body appear to need to do this as well, such as stem cells and sperm cells, because they need to replicate more than 50 times in a lifetime. However, it is probably too simple a view to assume that telomere shortening is the only major mechanism that causes aging of an organism but there is definitely a need to study the fate of telomeres and telomerase during human aging to further establish or clarify their role in the aging process and the development of diseases.Is the solution to the need for telomere assays the HT Q-FISH Assay? The answer appears to be yes.The HT Q-FISH assay developed by Canela at al. in 2007 uses an automated HT Q-FISH telomere-length ysis platform. This HT Q-FISH method builds on conventional Q-FISH and combines labeling of telomeres in interphase nuclei with a fluorescent nucleotide probe, here a peptide nucleic acid (PNA) probe, against telomeric repeats, with automated HT microscopy in 96-well plates. The researchers showed that with this procedure, an entire 96-well plate can be processed in about 2 hours. Validation of the method was done with a panel of mouse and human cell lines by determining their telomere lengths either by conventional telomere Q-FISH on metaphase spreads or HT Q-FISH.  This assay is a further development of the general Q-FISH method.FISH probesFor the staining of chromosomes labeled oligonucleotide probes directed against highly conserved mammalian telomere repeat sequences [5’-CCC TAA CCC TAA CCC TAA-3’; Telomere Probe] and against mouse major satellite repeats [5’-TCG CCA TAT TCC AGG TC-3’; Centromere Probe] were used. In recent years popular probes of this type are PNA based and labeled with Cy3 or FITC at the 5’ or 3’ end.  Another probe, that targets the (TTAGG)4 repeat can also be used to detect and stain  highly conserved repeats consisting of (TTAGGG)n and (CCCTTA)n sequences in every human chromosome.  Human chromosomes contain stretches of up to 30,000 C and G bases repeating over and over that often occur adjacent to gene-rich areas, forming a barrier between the genes and the non-coding DNA that used to be called “Junk DNA.”  In general FISH probes are targeted towards repetitive sequences present in the chromosomes. Scientists now believe that CpG islands help to regulate gene activity and more evidence has been accumulated in recent years to support the functional significance of satellite DNA sequences.A list of selected fish probes: These types of probes are usually labeled with Cy3 or FITC at the 5’ or 3’ end and artificial nucleic acids such as BNAs, LNAs and PNAs are incorporated in the oligonucleotide sequence. Many more probes can be designed to allow for selective chromosome painting.Workflow of the HT Q-FISH MethodInstruments for Bioimaging Confocal microscopes (left) and the pathways of the light beams are shown (right).Bioimaging of the plates or slides can be done with confocal instruments such as the “BD Pathway Bioimaging Systems” or any other similar confocal microscope may they be from BioRad, Nikon, Leica and Zeiss or any other instrument provider.A note to the readers of this blog: If you think I am biased towards a specific instrumentation provider please e-mail me your information for the instrument and the FISH application and I will add to this blog it as well {klaus_linse@biosyn.com}.Conventional or general Q-FISHThe conventional or general Q-FISH method uses fluorescent molecules to vividly paint genes or chromosomes and is particularly useful for gene mapping and to identify chromosomal abnormalities. The method involves the preparation of metaphase-arrested cells after adding colcemid to the culture medium. Colcemid disrupts microtubules in mitotic cells and arrest the cells in the metaphase state. Next, the cells are trypsinized and resuspended in a hypotonic buffer where the cells swell and the chromosomes spread. In the next step, the cells are fixed by removing the hypotonic solution using a centrifuge and are resuspended in a methanol/glacial acetic acid fixative. Slides for microscopy are prepared by placing a few drops of the cell suspension onto a microscope slide and let air dry overnight. The following day the slide is immersed in PBS for several minutes. Next, the slides are transferred into a 4% formaldehyde solution and fixed for several minutes, followed by washing steps with PBS buffer. The slides are then further processed with pepsin to digest proteins and peptides. Hybridization is done with a fluorescently labeled probe, usually a PNA probe that is labeled with a fluorophore (Cy3 or FITC), by placing a small volume of the hybridization mixture onto a cover slip and then onto the microscope slide which contains the fixed cells. However, other types of probes that target the same genetic regions may be used as well. Next, the DNA is denatured with a heat treatment using a preheated oven in which the chromosomal DNA in the cell is denatured at 80°C for several minutes. In the following step the slide is then left at room temperature for several hours to allow the probe to hybridize to complimentary DNA. Next, the slides are washed to remove the unbound probe and the DNA is counterstained with DAPI or PI and the microscope mounting medium is then placed onto the cells. This medium generally contains DAPI and an antifade solution to preserve the PNA fluorescence and reduce photobleaching. DAPI or 4′,6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA whereas propidium iodide (PI) is a fluorescing intercalating reagent (Mw: 668.4 Da) that also binds to nucleic acids. When bound to nucleic acids, its excitation maximum is 535 nm and its emission maximum is 617 nm. The ysis of stained samples is done by “Image Capture” where fluorescent reference beads are imaged first in order to ensure the proper set-up of the camera and fluorescent microscope used. A metaphase cell is then manually selected and centered for the camera. Two types of images are taken: pictures of the stained chromosomes in their metaphase state and fluorescent images of the telomeres. Superimposing the two images generates a combined image. The resulting image allows to karyotype or assign nomenclature to the stained chromosomes. Furthermore, the intra-chromosomal distribution of telomere length in p-arms (the short arms) versus q-arms (long arms of a chromosome) can also be measured. Normalizing the fluorescence intensities allows comparison of data from different experiments. Plasmids with a known number of telomeric repeats can be used as standards to help relate telomere fluorescence and telomere length.  The following picture shows the general workflow of the method.General workflow of the FISH method[Source: http://en.wikipedia.org/wiki/Q-FISH%5DThe principles of the Fluorescence In Situ Hybridization (FISH) method are explained at the following website:http://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327%5D.A research group in Germany in 2001 reported that they had developed a then new multicolor-FISH approach they called centromere-specific multi-color FISH (cenM-FISH). This multicolor FISH technique allows the simultaneous characterization of all human centromeres by using labeled centromeric satellite DNA as probes. Rapid identification of all human centromeres is achieved by assigning them to their individual pseudo-color in one single step. The researchers argue that this method is a powerful tool in molecular cytogenetics. CenM-FISH uses whole chromosome painting (WCP) probes and distinguishes all centromeric regions apart from the evolutionary highly conserved regions on chromosomes 13 and 21. Furthermore, this method is useful for prenatal, postnatal, and tumor cytogenetic screening as well as for the identification and characterization of heterochromatic material inserted into homogeneously staining regions.More recently, in 2012, researchers at the Harvard Medical School and the University of Michigan published a paper in which they described the design and synthesis of a platform useful for the visualization of genomes with oligopaint FISH probes (Beliveau et al.).  This technique, called “Oligo Paint Fish Platform“, allows a scientist to study any sequenced organism by interrogating the relationship between nuclear architecture or chromosome position and processes such as gene expression by its ability to visualize chromosomes in situ.  The method combines an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) with a bioinformatic platform. The proper design of the probes enables the technology to be used for the study of any organism whose genome has been sequenced. The method gives researchers a precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases. The researchers anticipate that this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes in the future.Outline of the Oligo Paint FISH Platform(Source: Beliveau et al. 2012)In conclusion, it is anticipated that the recent increase in activities to design and synthesize new types of artificial nucleic acids, such as BNAs, will allow the design of more sensitive and specific FISH probes in the future that. This will lead to an exponential increase in biological information which hopefully will also translate into the design of better and smarter drugs or treatments for human diseases with less side effects.Further links to review are:http://www.biosyn.com/TEW/FISH-glossary-Fluorescence-in-situ-hybridization-technique.aspxhttp://downsyndrome.about.com/od/diagnosingdownsyndrome/a/FISHarticle.htmhttp://www.ornl.gov/sci/techresources/Human_Genome/project/journals/insights.shtmlReferencesBeliveau BJ, Joyce EF, Apostolopoulos N, Yilmaz F, Fonseka CY, McCole RB, Chang Y, Li JB, Senaratne TN, Williams BR, Rouillard JM, Wu CT.; Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21301-6. doi: 10.1073/pnas.1213818110. Epub 2012 Dec 11.Andrés Canela, Elsa Vera, Peter Klatt and María A. Blasco; High-Throughput Telomere Length Quantification by FISH and Its Application to Human Population Studies. Proceedings of the National Academy of Sciences of the United States of America Vol. 104, No. 13 (Mar. 27, 2007), pp. 5300-5305. Published by: National Academy of Sciences, Article Stable URL: http://www.jstor.org/stable/25427190Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985; 43:405-13.Greider CW, Blackburn EH. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 1989; 337:331-7.Hayflick L, Moorhead PS (1961). “The serial cultivation of human diploid cell strains”. Exp Cell Res 25 (3): 585–621. doi:10.1016/0014-4827(61)90192-6. PMID 13905658.Hayflick L. (1965). “The limited in vitro lifetime of human diploid cell strains”. Exp. Cell Res. 37 (3): 614–636. doi:10.1016/0014-4827(65)90211-9. PMID 14315085.Peter M. Lansdorp, Nico P. Verwoerd, Frans M. van de Rijke, Visia Dragowska, Marie-Térèse Little, Roeland W. Dirks, Anton K. Raap and Hans J. Tanke; Heterogeneity in telomere length of human chromosomes.  Human Molecular Genetics, 1996, Vol. 5, No. 5 685–691.Prosser J, Frommer M, Paul C, Vincent PC.; Sequence relationships of three human satellite DNAs. J Mol Biol. 1986 Jan 20;187(2):145-55.Shay, J. W. and Wright, W. E. (2000). “Hayflick, his limit, and cellular ageing” (PDF). Nat. Rev. Molec. Cell Biol. 1 (1): 72–76. doi:10.1038/35036093.Szostak JW, Blackburn EH. Cloning yeast telomeres on linear plasmid vectors. Cell 1982; 29:245-255.Ugarkovic D.; Functional elements residing within satellite DNAs. EMBO Rep. 2005 Nov;6(11):1035-9.Nietzel A, Rocchi M, Starke H, Heller A, Fiedler W, Wlodarska I, Loncarevic IF, Beensen V, Claussen U, Liehr T. A new multicolor-FISH approach for the characterization of marker chromosomes: centromere-specific multicolor-FISH (cenM-FISH). Hum Genet. 2001 Mar;108(3):199-204.Categories: Aging, Aging Theories, Artificial Nucleic Acids, Bioysis, Bioinformatics, BNA RNA, BNAs, Bridged Nucleic Acid, Bridged Nucleic Acids, Cancer, Cancer Risk, Centromere, Chromatin, Chromosome Painting, Comparative Genomics, DNA, DNA ysis, DNA Editing, DNA Hybridization, DNA Structure, DNA Synthesis, DNA-BNA chimeras, FISH, FISH Probes, Gametogenesis, Gene Editing, Gene families, Genetics, heterochromatin, Human Genetics, Hybridization, Karyotyping, Leukemia, LincRNA, lncRNA, Long noncoding RNA, Melanoma, miRNA, miRNA regulation, Molecular Probes, non-coding RNAs, Oligonucleotide Synthesis, piRNA, Regulatory RNA, RNA, RNA Editing, RNA FISH, RNA Structure, RNA Synthesis, RNA World, RNAi, Synthesis, Telomere, TelomersTags: Aging, Cancer, Custom Long RNA, DNA, DNA ysis, genome, Long RNA Synthesis, miRNA, Oligo Synthesis, PCR, probes, RNA, TelomersFill in your details below or click an icon to log in: You are commenting using your WordPress.com account. 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