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FISH slides 2011 - Karyotyping Which chromosomes are there Are they normal Tools of the trade Gene identification by breakpoint

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Unformatted text preview: Karyotyping Which chromosomes are there? Are they normal? Tools of the trade Gene identification by breakpoint Changes in chromosome structure and number by FISH Fluorescence In Situ Hybridization Old style banding allows identification of each chromosome by bands and size http://www.sciencemuseum.org.uk/exhibitions/genes/154.asp Chromosome painting and banding allows identification of each chromosome Spectral karyotyping (SKY) is a laboratory technique that allows scientists to visualize all of the human chromosomes at one time by "painting" each pair of chromosomes in a different fluorescent color. Specific color pattern for each chromosome One color per chromosome www.genome.gov/10000208 http://www.bio.unc.edu/courses/2003spring/biol050h/biolog2.jpg Interphase vs metaphase Interphase vs metaphase probes on same chromosome are close DOTS vs BANDS 3 sep probes INTERPHASE IS CHEAPER AND EASIER vs colchicine-arrested metaphases 2 probes on same chromosome are close to each other 1 Detecting Aberrations A translocation break (t4:?) 1 Detecting aberrations 1 2 2 t4 t4 4 Abberations and FISH Chromosome painting, or fluorescence in situ hybridization (FISH) Figure. An example of FISH-treated metaphase chromosomes Here, chromosomes 1, 2, and 4 were labeled yellow with FISH and the other chromosomes were stained red. Translocations between yellow and red chromosomes are detected. The left picture represents a normal cell (the numbers in the figure indicate chromosome numbers) and the right picture is an example of reciprocal translocation with two bi-color chromosomes (indicated by two arrows). www.rerf.or.jp/ Gene/eng/fig/fishf.htm A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation Nature Genetics 24, 167 - 170 (2000) INVERSION Williams-Beuren Syndrome (WBS) (OMIM #194050) = very interesting. Inversions, deletions, Lenhoff, contiguous gene, autism etc. . 3 probes on same chromosome Figure 1. Genomic region of the X chromosome containing the X;2 translocation breakpoint and TM4SF2 (Xp11.4 tetraspanin). a, Physical map of the Xp11.4 locus and genomic structure of TM4SF2. The position of the breakpoint is indicated by a vertical dotted line. The eight exons are indicated (filled boxes) and their mapping showed that TM4SF2 spans at least 130 kb. STSs and ESTs are indicated (top) and the order of ESTs that map in the overlapping region between YAC clones 770D12 and 957F5 was defined here through their position on the BAC contig. EST AA9113249 was reported in the EST database, but its position in this region was deduced through sequencing of the ends of BAC clone 110L03. All BAC clones were from the CEPH B 751 BAC library. b, Metaphase spread from the patient with the X;2 translocation showing FISH signals obtained with BAC clone 107D22 (red) and with a chromosome-X−specific probe (green). Chromosomes X, derX and der2 with hybridizing signals are indicated. Note the hybridization signals on both derX and der2 chromosomes, indicating that this BAC clone spans the breakpoint 107D22 R B W W B R B W R R W B X cen tel cen 2 cen Inversion Normal X cen Fig 1: Inversions of chromosome 7 in Williams-Beuren syndrome. Bottom three dots show normal order: green, yellow, pink. Top three dots show WB inversion: yellow, pink, green. http://www.genomenewsnetwork.org/articles/04_03/chrom7.shtml http://www.utoronto.ca/osborne/research.html acute non lymphoblastic leukaemia (ANLL) YOUR PROBE SPANS AN INV BREAK Another inversion Williams-Beuren Syndrome (WBS) (OMIM #194050) B R R W B W W B R W R B inv(16)(p13q22) G- banding (left) - Courtesy Jean-Luc Lai and Alain Vanderhaegen; ; R- banding center below: - Courtesy Christiane Charrin, center top and FISH (right) -Courtesy Pascale Cornillet-Lefebvre and Stéphanie Struski. Commercial FISH probe, split in the inv (16) tel cen Chr 7 cen cen Fig 1: Inversions of chromosome 7 in Williams-Beuren syndrome. Bottom three dots show normal order: blue, white, red. Top three dots show WB inversion: white, blue red. http://www.genomenewsnetwork.org/articles/04_03/chrom7.shtml http://www.utoronto.ca/osborne/research.html http://atlasgeneticsoncology.org/Anomalies/inv16.html 2 Detecting aberrations & environmental causes of chromosome breaks Abberations and FISH Southern blots Hexavalent chromium! Chromate treated lung cells •  When double strand breaks form, Histone gamma-H2AX (H2AX) rapidly accumulates on them and labels the break for repair. •  An H2AX-specific fluorescent antibody, appears as a glowing green dot. •  Each dot represents a double strand break in the DNA untreated lung cells •  A breeding ground for Dfs, Inv, Translocations http://www.usm.maine.edu/toxicology/research/chromium.php Hexavalent chromium! Gel electrophoresis Hexavalent chromium! DNA from Chromate treated lung cells •  Comet indicates trailing broken DNA fragments Electrophoretic migration untreated lung cells Figure 6B. A spectral karyotype of the metaphase in figure 6A. This karyotype shows a translocation between chromosomes 2 and 5. http://www.usm.maine.edu/toxicology/research/chromium.php http://www.usm.maine.edu/toxicology/research/chromium.php Chronic exposure to metal ions from early metal-on-metal hip bearings may result in chromosomal aberrations, according to recent findings, but the consequences of these aberrations remain unknown. Orthopedics today 2006 Are these findings of clinical significance? We have absolutely no idea, said Edward Dunstan, FRCS, of Stanmore, England. Our patients are otherwise well. t How do you know when you have the DNA that contains the gene of interest? X t X Detecting specific genes by probes breakpoint spanning probes. X This image of abnormal chromosomes shows translocation between chromosomes 2 and 15 and aneupoidy loss of chromosomes 9, 16 and 17. This fluorescent in situ hybridization chromosome painting technique shows aberrations in peripheral leucocytes in a male patient with a metal-on-metal implant of 38 years duration. http://www.orthosupersite.com/view.aspx?rid=19445 FISH Southern blots 3 Aneuploidy screening (fig.2): Using probes for chromosomes 13, 18, 21, X and Y, to rapidly screen samples of amniotic fluid for the common trisomies. Results usually available in 48 hours. PATIENT NORMAL CONTROL Detecting changes in chromosome # Could have other interpretations, e.g. inversion or translocation break. Hence the value of internal control probes. www.humangenetics.org.uk/ molcyto.htm CENTROMERE PROBES Trisomy 18 Trisomy 8 (+8). FISH. Centromere probe for chromosome 8 (green). Three cells each with trisomy 8(three green signals each) (interphase preparation) http://www.accessmedicine.com/search/searchAMResultImg.aspx?rootterm=trisomy+x+syndrome&rootID=42751&searchType=1 http://www.biomedcentral.com/content/figures/1471-2407-6-249-2.jpg 3 copies of both chr 21 probes - conclusion – trisomy 21 Use 2 probes in different parts of chr 21 to distinguish trisomy from abberation [or a centromere specific probe] Chromosome painting lights up the extra chromosome 21 in the cells of people with Down syndrome (pink and light blue dots). Joyce Harper/Wellcome Photo Library http://www.sciencemuseum.org.uk/exhibitions/genes/156.asp CENTROMERE PROBES Trisomy 8 (+8). FISH. Centromere probe for chromosome 8 (green). Three cells each with trisomy 8(three green signals each) (interphase preparation) http://www.accessmedicine.com/search/searchAMResultImg.aspx?rootterm=trisomy+x+syndrome&rootID=42751&searchType=1 4 Small duplications Micro deletions Fluorescence in situ hybridization (FISH) using wcp11 to characterize a cryptic insertion into the long arm of one chromosome 2. http://images.google.com/imgres?imgurl=http://glccontario.tripod.com/sitebuildercontent/ sitebuilderpictures/fish.jpg&imgrefurl=http://glccontario.tripod.com/ &h=266&w=328&sz=9&hl=en&start=13&tbnid=UILj4G-0_jEj5M:&tbnh=96&tbnw=118&prev=/images%3Fq %3Dfish%2Bin%2Bsitu%2Bhybridization%26gbv%3D2%26svnum%3D10%26hl%3Den%26sa%3DG Summary Microdeletion analysis (fig.1): Used in special cases where the referring clinician suspects a specific syndrome. These are often not detectable by conventional cytogenetic methods. Syndromes for which a FISH test is available include: Williams, Prader-Willi, Angelman, Miller-Dieker, Smith-Magenis, Di George. Fig 1: FISH detects a deletion of the elastin gene on chromosome 7, in a patient with Williams syndrome www.humangenetics.org.uk/ molcyto.htm How many ways could this be interpreted? 2 probes for 2 different chromosomes •  Probes can be used to identify changes in gene or chromosome number, or abberations. •  The level of resolution = the size of the probe. •  Southern blots interrogate very defined domains •  FISH interrogates larger domains Trisomy, translocation, inversion, Possible duplication of the red 5 ...
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This note was uploaded on 12/13/2011 for the course BIOSCI 137 taught by Professor Staff during the Fall '11 term at UC Irvine.

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