Cosmid probes

For these reasons, it has been difficult to develop procedures that allow cosmid probes to be detected reliably in whole yeast cells. Other investigators have demonstrated that such probes may be used if the cells are subjected to heavy extraction with detergents, proteases, and other agents, effectively spreading the yeast nucleus onto microscope slides. However, to attain the goal of whole-cell preservation has required a different approach. 

For repetitive DNA sequences, such as alphoid repeats, especially for identification of centromeric regions on chromosomes, as well as in mterphase nuclei, I routinely prepare labeled probes by PCR using a centromeric DNA-specific set of primers. For single-copy gene mapping by FISH, it is essential to use DNA cloned into either cosmids or in yeast artificial chromosome vectors (YAC) m order to obtain the level of sensitivity that will enable visualization of signal with a fluorescence microscope... 

Fig. 2. Map of the Haloferax volcanii genome (top) the chromosome (bottom) the plasmids. Cosmids are represented by arrows, site-rich regions (oases) by heavy line, and markers placed by hybridization with unique probes are shown inside the circle (all from ref. [15]). Outside the circle are the results of probing with repeated sequences (ISH51, D), with labelled tRNA, 7S and ribosomal RNAs and (outermost) auxotrophic markers mapped by complementation. From ref. [86].

We chose Acetobacter xylinum JCM 7664 (equivalent to IFO 13693), which shows high activity of ceUulose synthesis. Parts of besA and bcsCwere amplified by PCR based on consensus sequences of A xylinum strains 1306-3 [1] and ATCC 53582 [2] and used as probes for initial screening of cosmid and plasmid libraries. 

Formamide hybridization mix For single-copy probes (e.g., cosmids), 2X SSC, 500 pg/rnL salmon sperm DNA (SSDNA), 10% dextran sulphate, 50% formamide. 

Cosmid clones Genomic sequences 45 kb Mapping Interphase cytogenetics Usually make good FISH probes High... 

Suppression hybridization (Section 12.1.2) may be required if the probe contains repetitive sequences (e.g., lambda, cosmid or YAC probes) which would hybridize much faster and give a stronger signal than the specific sequence. It is also recommended to use low salt concentrations (e.g., 1 x SSC). Double labeling can then avoid statistical analysis of many metaphase chromosomes (Lawrence et al., 1990). 

Chromosomal localization of the axl gene is done by fluorescence in situ hybridization (FISH). The biotin-labeled axl probe is from a cosmid vector containing a 41-kb genomic fragment. The slides with human metaphase cells for chromosomal DNA to be denatured are immersed into a solution of 70 % formamide-x4 in saline sodium citrate buffer (SSC) atpH 7.0 (0.15 M NaCl 0.015 M sodium citrate) at 70 °C for 2 min. For the slides, the hybridization mixture kept at 75 °C consists of 50 %formamide,xl SSC, 10 % dextran sulfate pH 7.0, and 150 mL unlabeled sonicated total human genomic DNA add 50-100 ng labeled probe. The mixture is incubated at 37 °C for 10 min. The slides are washed in 50 % formamide and SSC x3, 5 min each at 40 °C and in SSC x3 at 40 °C. The detection reagent consists of x4 SSC-0.1 % Triton, and 1 % bovine semm albumin for 3 washes 3 min each at 40 °C. The slides are incubated with fluorescein isothiocyanate-conjugated avidin at 37 °C for 30 min. Metaphase cells are counterstained with 4,6-diamidino-2-phenylindole dihydrochloride 200 ng/mL in 2x SSC for 5 min at... 

---