FISH - PROVIDING FISH AS A ROUTINE SERVICE DR TIEN SIM LENG...

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Unformatted text preview: PROVIDING FISH AS A ROUTINE SERVICE DR TIEN SIM LENG SENIOR CONSULTANT DEPARTMENT OF HAEMATOLOGY HEAD, CYTOGENETICS LABORATORY DEPARTMENT OF PATHOLOGY WHAT IS FISH ? WHAT IS A FISH ? FISH = Fluorescence In Situ Hybridization CYTOGENETICS + MOLECULAR GENETICS (Chromosome study) (DNA study) = MOLECULAR CYTOGENETICS (e.g .FISH study) FISH FISH METAPHASE FISH : DIVIDING CELLS INTERPHASE FISH : NON-DIVIDING CELLS WHY FISH ? UNDERSTANDING STANDARD CYTOGENETICS NORMAL CHROMOSOMES KARYOTYPING CHROMOSOME IDEOGRAM International System for Human Cytogenetic Nomenclature ISCN 1995 Number 1-23, X, Y Long arm q Short arm p Landmarks; ends of chromosomes, centromeres, characteristic bands Regions and bands are numbered consecutively from the centromere outward. 9q34 chromosome 9 long arm region 3 band 4 CHROMOSOMAL ABNORMALITIES I. NUMERICAL II. STRUCTURAL I. NUMERICAL ABNORMALITIES 1. HYPODIPLOIDY 2. HYPERDIPLOIDY 3. ANEUPLOIDY II. STRUCTURAL ABNORMALITIES 1. DELETIONS 2. INSERTIONS 3. DUPLICATIONS 4. TRANSLOCATIONS 5. REARRANGEMENTS DELETION AT LONG ARM OF CHROMOSOME INSERTION DUPLICATION RECIPROCAL TRANSLOCATION RING CHROMOSOME WHY FISH ? LIMITATIONS OF STANDARD CYTOGENETICS 1. Deletions smaller than a few million base pairs are not routinely detectable. 2. Chromosomal abnormalities with indistinct or novel banding patterns can be difficult or impossible to interpret. 3. Analyzable metaphases may not be always possible to obtain e.g. poor specimen, too few or no dividing cells, post-treatment specimen, some types of solid tumours. METAPHASE CYTOGENETICS INTERPHASE FISH * Labour and experience required *Relatively simple *Complete analysis requires days *Complete analysis in a few hours * Few cells are karyotyped *Hundreds to thousands of cells are evaluated *Successful growth of cells *Performed on non-dividing into metaphases cells *The whole genome is studied *Only few abnormalities HOW TO FISH ? FISH Each probe is specific to one region of a chromosome (pair), and is labeled with fluorescent molecules throughout it's length. Each microscope slide contains many metaphases. Each metaphase consists of the complete set of chromosomes, one small segment of which each probe will seek out and bind itself to. The first step is to break apart (denature) the double strands of DNA in both the probe DNA and the chromosome DNA so they can bind to each other. This is done by heating the DNA in a solution of formamide at a high temperature (70 C) Next, the probe is placed on the slide and a glass coverslip is placed on top. To prevent evaporation, the edges of the coverslip are sealed with rubber cement. The slide is then placed in a 37 C incubator overnight for the probe to hybridize with the target chromosome. Overnight, the probe DNA seeks out it's target sequence on the specific chromosome and binds to it. The strands slowly rejoin (reanneal). The next day, the coverslip is removed, and the slide is washed in a salt/detergent solution to remove any of the probe that did not bind to...
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This note was uploaded on 01/26/2012 for the course PDBIO 305 taught by Professor Woods,a during the Fall '08 term at BYU.

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FISH - PROVIDING FISH AS A ROUTINE SERVICE DR TIEN SIM LENG...

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