• Goldammer T, Rottengatter K, Weikard R, Brunner RM, Horstmann D, Gelhaus A, Hanotte O, Schwerin M (2004) Targeted generation of 16 sequence tagged sites for bovine chromosome region 5q21-q25 by microdissection. Chromosome Res 12: 309-315.
• Brunner RM, Sanftleben H, Goldammer T, Kühn C, Weikard R, Kata SR, Womack JE, Schwerin M (2003) The telomeric region of BTA18 containing a potential QTL region for health in cattle exhibits high similarity to the HSA19q region in humans. Genomics 81: 270-278.
• VAIMAN D, SCHIBLER L, OUSTRY-VAIMAN A, PAILHOUX E, GOLDAMMER T, STEVANOVIC M, FURET JP, SCHWERIN M, COTINOT C, FELLUOUS M, CRIBIU EP (1999). High-resolution human/goat comparative map of the goat polled/intersex syndrome (PIS): the human homologue is contained in a human YAC from HSA3q23. Genomics 56(1):31-9. 
• WEIKARD R, GOLDAMMER T, KÜHN C, BARENDSE W, SCHWERIN M (1997): Targeted development of microsatellite markers from the defined region of bovine Chromosome 6q21-31. Mammalian Genome 8:836-840.
  
• GOLDAMMER T, BRUNNER RM, SCHWERIN M (1997): Comparative analysis of Y chromosome structure in Bos taurus and Bos indicus by FISH using region specific, microdissected and locus specific DNA probes. Cytogenetics and Cell Genetics 77:238-241.
• GOLDAMMER T, WEIKARD R, BRUNNER RM, SCHWERIN M (1996): Generation of chromosome fragment specific bovine DNA sequences by microdissection and DOP-PCR. Mammalian Genome 7:291-296.

Chromosome microdissection
All chromosome manipulations were observed under an inverted microscope (Sedival, Carl Zeiss Jena) equipped with a rotating slide table. Microdissection of chromosome fragments was performed with glass microcapillaries (Clark Electromedical Instruments, England) which were fixed and controlled by a motordriven micromanipulator 3K (Märzhauser, Germany). Microcapillaries were forged with a pipette puller (Model 750, Clark Electromedical Instruments, England) to produce the correct tip diameter for the dissection of the chromosome fragment of interest. The range of the capillary which contact the slide determined the size of the dissected chromosome area. In order to avoid DNA contamination, all microneedles were fresh prepared for each dissection and treated with ultra violet light for a few minutes prior to use.

Topoisomerase I treatment
Dissected chromosome material adherent to the tip of a microneedle was transferred into a 0.2 ml tube containing 5µl of 40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 50 mM NaCl, 200µM each dNTP, 1 unit Topoisomerase I (Promega), and 1 µl sequenase reaction buffer (pH 7.5) (Guan et al., 1993), and 10 pmol of the degenerate oligonucleotide primer 6-MW 5'- CCGACTCGAGNNNNNNATGTGG-3' (Telenius et al., 1992). The drop was incubated at 37°C for 30 min, followed by a denaturation step at 96°C for 10 min.

PCR amplification
Amplification of chromosome fragments was carried out basically as described by Guan et al. (1993). In detail, directly after topoisomerase I treatment and an initial denaturation step of 5 min at 94°C, eight cycles of Sequenase-PCR with a denaturation step at 94°C for 1 min, an annealing step at 30°C for 1 min, and an extension step at 37°C for 1,5 min was performed by the addition of approximately 0.4 units of T7 DNA polymerase (Sequenase version 2.0, USB) at each annealing step. Thus 0.25 µl T7 DNA polymerase (1.6 U/µl) was added to the 5 µl reaction mixture in each cycle.
After these initial preamplification cycles a PCR reaction catalyzed by Taq DNA polymerase (Perkin Elmer Cetus) was performed without delay in the same reaction tube. The final volume of the PCR mixture was 50 µl (including the 7 µl reaction mixture) containing a final concentration of reaction mixture with 200µM of each of the four dNTP's, 50 pmol DOP-6MW primer, 3.5 mM MgCl2, 14 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM NaCl, 0.1 mg/ml Gelatine, and 2 U Taq DNA polymerase.
After an initial denaturation at 94°C for 4 min, 35 cycles of DOP-PCR were carried out with denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1,5 min. The final extension time was increased to 5 min. PCR products (8 µl) were characterized by gel electrophoresis on a 1% agarose gel.
Biotinylation of amplified microdissected Darried out as described above using 2 µl of the first PCR reaction and 20 µM Biotin-16-dUTP (Boehringer). The reaction tubes were heated to 94°C for 4 min followed by 16 cycles at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 3 min, and a final extension for 5 min. All PCR reactions were performed oil free in a Hybaid-Cycler.

Fluorescence in situ hybridization (FISH)
Biotinylated DNA probes for FISH were precipitated with 3 v/v 100% Ethanol and 0.1 v/v 3M Na-acetate (pH 6.8) at -80°C overnight. Hybridization of probes to bovine chromosomes was performed essentially as described by Pinkel et al. (1986, 1988). A DNA mixture containing 150 to 300 ng of the probe, 1-5 µg bovine Cot-1 DNA and 1-5 µg salmon sperm DNA was prepared, vacuum dried and then suspended in 2 µl of deionized water and 8 µl of hybridization mixture for 1 to 2 hours. Hybridization mixture containing 50% formamide (Fluka No. 47671), 10% dextransulphate and 2xSSC is stable stored at -20°C for several month. The DNA-hybridization mixture was denatured at 75°C for 8 min followed by a preannealing step for chromosome in situ suppression at 37°C for 1.5 to 3 hours in an DNA Thermal Cycler (Landegent et al., 1987; Lichter et al., 1988; Lengauer et al., 1991). Slides with digitized GTG-banded metaphase spreads were incubated on a hot plate, overlaid with 100 µl 70% formamide, 2xSSC, covered with cover slips and denatured for 4 min at 80°C. After this procedure the slides were quickly transferred to a Coplin jar containing ice-cold 70% ethanol and agitated to rinse off the denaturing solution. Slides were dehydrated serially in ethanol (80, 90, 100%) for 3 min each and dried with an air jet. The preblocked DNA-probes were then placed on prewarmed (37°C) slides and covered with 18 x 18 mm cover slips. Cover slips were sealed with rubber cement and slides were incubated in a moist chamber at 37°C for 16 to 20 hours to complete the DNA hybridization.

Probe detection
After hybridization, the cover slips were removed gently, and the slides were washed three times in 50% formamide (Fluka 47670), 2xSSC at 45°C for 3 min and three times in 2xSSC at 45°C for 2 min each. The slides were then blocked for 30 min in PNM-buffer [0.1 M NaH2PO4, 0.1 M Na2HPO4 (pH 8.0), 0.1% Nonidet P-40; 5% nonfat dry milk; 0.02% sodium azide] at ambient temperature to reduce nonspecific antibody binding.
In order to visualize the probe slides were overlaid for twice with 100 µl fluorescein isothiocyanate (FITC) conjugated to avidin (Vector Laboratories) in PNM (3:1000) at 37°C covered with cover slips and incubated at 37°C for 40 min. The signal was amplified using one layer of 100 µl biotinylated antiavidin antibody (Vector Laboratories) in PNM (11:1000) at 37°C for 30 min. Slides were washed three times between incubations in 0.1 M PN-buffer [0.1 M NaH2PO4, 0.1 M Na2HPO4 (pH 8.0), 0.1% Nonidet P-40, 0.02% sodium azide] for 2 min each at ambient temperature.
Chromosome material was counterstained with 200 µM propidium iodide, 2xSSC for 4 min and rinsed in deionized water for 1 min. Finally slides were overlaid with glycerol containing 2.3 % DABCO (1.4-diazabicyclo-[2.2.2]octane; Sigma) as antifade solution and covered with a cover slip.
Metaphase spreads were analyzed with a Nikon Microphot FXA microscope equipped with a specific band pass filter BP475 (Carl Zeiss Jena) for fluorescein signals. Photographs were taken with Kodak Gold 400 film or alternatively, images were digitized with MacProbe FISH software (PSI).
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