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Ref: Cytometry2001, Vol 43(2), p101-109.Get article in PDF format here
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Slide and probe denaturing can be performed together (called "simultaneous or direct denaturing") or separate (called "separate or indirect denaturing"). Numerous experiments with both commercial and custom prepared probes, from plasmids or cosmids to complex microdissected or painting probes showed that results were similar with both procedures. The main difference was that the simultaneous denaturing protocol (which is shorter) required a larger amount of competitor DNA (Cot1-DNA).
- In the simultaneous denaturing process, the slide can be placed directly on a heated metal block for 2 minutes at 72-80° C. This procedure benefits from a longer chemical aging process (1-2 minutes instead of 10-15 seconds) to preserve chromosome morphology. In this lab, we are routinely denaturing slides on the metal block of a thermocycler. The block is programmed to (1) increase gradually (90 seconds) the temperature to 75° C, (2) hold the temperature 90 seconds at 75° C and (3) to gradually (90 seconds) decrease the temperature back to the room value. This gradual heating and cooling is gentler on the chromosomes and requires just a few seconds of aging in ethanol prior to denaturing. Chromosome architecture is preserved better and hybridization is very efficient.
- In the separate denaturing protocol, slides and DNA probes are separately denatured. Slides are denatured in 70%formamide(FA)/2xSSC in a Coplin jar at 75° C, followed by rinsing, 3 minutes each in 70% and 100% ethanol at room temperature. The use of ice cold ethanol should be avoided, as sudden temperature changes affect chromosome morphology. Gradual heating and cooling of the slide can be applied to the separate denaturing process as well, in one of two ways. (1) 150-200ul 70%FA/2xSSC are pipetted directly on the slide, covered with a coverslip and the slide is denatured gradually on the metal block of a thermocycler (as described). The coverslip is removed, and the slide is rinsed in 70% and 100% ethanol, air dried and used. (2) Three jars with 70%FA/2xSSC solution are heated at 45° C, 60° C and 75° C, respectively. Slides are kept 5-10 seconds each in the 45° C and 60° C solutions, then incubated 90-120 seconds at 75° C and kept again 5-10 seconds each in the 60° C and 45° C solutions. Denaturing is followed by brief rinsing in 70% and 100% ethanol and air drying. This denaturing approach is gentler on chromosome morphology and eliminates the need of a thermocycler, for laboratories not equipped with one.
Regardless of the denaturing protocol, it is best that ethanol solutions used for rinsing slides are not chilled, but kept at room temperature. This prevents exposing the slides to large temperature variations. We noticed that, placing slides directly into the hot formamide solution (75° C), or from this solution directly into ice-cold ethanol, results in distorted chromosome morphology without improved denaturation. Gradual heating and cooling proved just as efficient, and preserved chromosome morphology much better. The reason DNA remains denatured after gradual cooling is that formamide numerous breaks into the DNA. It is at these breaks that the two strands of DNA uncoil and stay denatured. Simple fiber-stretching experiments or fiber FISH can prove this phenomenon.
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Last modified on: Feb12, 2001