Chromosome Microdissection Using Conventional Methods
Chromosome Microdissection Using
Conventional Methods I. INTRODUCTION During cytogenetic analysis there are frequently complex chromosomal structural aberrations, which are unidentifiable by chromosome banding analysis. Fluorescence in situ hybridization (FISH) combined with G-banding analysis (Xu and Wang, 1994) has significantly improved the accuracy of chromosome identification. However, the efficacy of FISH analysis depends on the probes chosen, which in turn depend on the G-banding result. It is very difficult to choose the adequate probe(s) for FISH analysis when no clue is available from banding analysis. Therefore, FISH can serve as a tool for verification and confirmation. The recently developed multicolor or spectral karyotyping highly facilitates the identification of complex interchromosomal structural abnormalities. However, its application is limited to nonhomologous rearrangements and its efficacy depends on the size of the chromosome segment involved. Furthermore, it can identify the chromosomes involved but not the specific segment or breakpoints involved. In contrast, the chromosome microdissection approach provides a straightforward method for identifying any chromosomal segment of unknown origin by dissecting the segment of interest and isolating, amplifying, fluorescence labeling, and reverse in situ hybridizing DNA to normal metaphase spreads (Ludecke et al., 1989; Bohlander et al., 1992; Carter et al., 1992; Meltzer et al., 1992; Ruano et al., 1992; Telenius et al., 1992; Guan et al., 1993; Zhang et al., 1993). Once the components of the aberrant chromosome are identified, conventional FISH with whole chromosome painting probes can be applied to define the relative position of the components in the marker chromosome. This micro-FISH approach has been applied successfully in the identification of ring chromosomes (Xu et al., 1995, 1998), homogeneous staining regions (Xu et al., 1996; Abeysinghe et al., 1999), and double minutes (Sen et al., 1994). Thus, micro-FISH can be used to identify not only the origin of the marker chromosome, but also the regions and breakpoints. However, in the reverse FISH the specificity of the probe generated relies on the accurate recognition and dissection of the specific target chromosome/segment and on the precise extraction, amplification, and labeling of the DNA with minimum background contamination. The skills, procedures, and setup involved in micro-FISH are too complicated to be established in the routine clinical cytogenetic laboratory. This is the major limitation to the application of reverse FISH by chromosome microdissection. With a modified fixation procedure (Xu et al., 1995), however, specimens can be stored and shipped to experienced laboratories or diagnostic centers. In addition to its clinical application, chromosome microdissection can be used to generate band-specific probes (Guan et al., 1993) that can be used in establishing intrachromosomal spectral karyotyping. A combination of interchromosomal spectral karyotyping with intrachromosomal spectral banding analysis would be ideal for chromosome analysis in neoplasia. II. MATERIALS AND INSTRUMENTATION A. Materials Methyl alcohol (Fisher Scientific, #MD3017-4) Potassian chloride solution 0.075N (Irvine Scientific, #9281) Acetic acid, glacial ACS (Fisher Scientific, #A38-500) Colcemid 10µl/ml in HBSS (GIBCO, #15210-040) Trypsin (1:250) (GIBCO, #27250-018) KaryoMax Giemsa stain (Lab Chem Inc., #2C 14840-7) Hank's balanced salt solution ×1; without CaCl2 + Mg (Sigma, #119349) Buffer tablets "GURR" (GIBCO, #10582-013) Deionized distilled (dd)water T7 DNA polymerase (Sequenase, Version 2.0, USB #70755Y) Topoisomerase 1 (Promega, #M2851) Biotin-16-dUTP (Boehringer Mannheim, #1093070) Taq DNA polymerase (Applied Biosystems, #4311816) Universal primer 5'-CCGACTCGAGNNNNNNNATGTGG-3' (Telenius et al., 1992) 15-ml sterile polystyrene tubes (Corning, #43005) Microneedle (Sutter Instrument Co., #BR100-15) Coverslips (VWR, 24 × 60mm #48393-106) B. Instrumentation Incubator, 37°C Inverted microscope (Nikon DIAPHOT-TMD) 9-in. Pasteur glass pipettes (VCR, #53283-915) and bulbs Micromanipulators (Narashige, Models MM-88 and MO-302) Micropipete puller (Narishige, Model PB-7) Thermal cycler (MJ Research Inc., Model PTC-200) Slide warmer (VWR) Coplin jars (0.50-ml capacity) Thin-walled microtubes (Laboratory Products Sales, #430) Microcon YM-100 columns (Millipore, #42413) III. PROCEDURES A. Preparation of Cells for Microdissection This procedure is modified from those of Wang and Federoff (1972) and Xu et al. (1994). Solutions
Steps
B. Chromosome Microdissection Combinded with FISH Analysis (Micro-FISH) The procedure of micro-FISH is performed according to Guan et al. (1993) with modification (Xu et al., 1995). Solutions
1. Chromosome Microdissection and DNA Amplification Steps
Steps
3. Verification of the Specificity of the Dissected Chromsome/ Segment Steps
4. Identification of the Origin of the Dissected Chromosome/Segment(s) Steps
5. Forward FISH Analysis to Identify the Relative Position of the Components of the Dissected Chromosome Steps
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