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Topics: 1.Preparation 2.Aging 3.Denaturing 4.Methods 5.Table | Fig1 | Fig2 |
FISH: simultaneous vs. separate denaturing.
Numerous experiments with plasmids, cosmids and painting probes showed that both procedures yielded similar FISH results. The simultaneous denaturing protocol is shorter and requires a larger amount of competitor DNA (2-3x more) to block repetitive sequences, as there is no "preannealing" step. The chromosomes become somewhat thicker (puffier), primarily because of the increased viscosity of the hybridization buffer, which usually contains dextran-sulfate. To prevent chromosome distortion, final dextran sulfate concentration should be between 5-8%. The shape of the chromosomes also depends on the brand of formamide used (Fig. 2a-c). In our hands, the best formamide for simultaneous denaturing procedure was from Fluka, Milwaukee, WI. The slide/coverslip assembly can be denatured directly on a simple, heated metal block for 2 minutes at 75Ú C. However, gradual heating and cooling using a thermocycler was gentler, and preserved chromosome architecture better (superior DAPI banding). In the more common, separate denaturing protocol, slides are denatured separately from the labeled DNA probe, using a similar gradual heating/cooling scheme as described. Viscosity of the 70% formamide solution prevents water from evaporating, so there is no need to cover the slide/coverslip assembly. Please note that for the separate denaturing protocol, there were no visible differences among the brands of formamide tested. Regardless of the denaturing protocol, ethanol rinse solutions should be kept at room temperature, to prevent exposing the slides to large thermal shocks, which are detrimental to the chromosomal shape.
DAPI banding.
Preservation of chromosome morphology depends on the correct balance among three factors: type of aging, pepsin treatment and denaturing technique. Dry heat aging provides sharper DAPI bands but decreased hybridization signals compared with chemical aging. Gradual denaturing helps correct this problem, by allowing fresh, "soft" chromosomes to be denatured while preserving their architecture well (Fig. 2g-h). Pepsin treatment, while improving the hybridization results, always alters chromosome architecture and, therefore, the exposure time to the enzyme needs to be adjusted according to the aging and denaturing techniques. A useful "trick" for better DAPI banding on chemically aged slides is to incubate them for a few seconds in 1% formaldehyde or paraformaldehyde, immediately after pepsin treatment. Even 5-10 seconds are sufficient. Longer incubations, 30—60 seconds, yield better bands but visibly alter hybridization efficiency.
Conclusion.
Appropriate chromosome spreading technique, followed by the use of a short aging process, appropriate protease pretreatment and gradual denaturing yielded best hybridization results.