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DNA fibers
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Length measurements of DNA probes.
DNA stretching used in this laboratory is a 15-20 minutes procedure, which can be used to calculate the length of probes from 10 kb to hundreds of kilobases. Briefly, isolated DNA of any probe is diluted to 1-5ng/ul in 10 mM AMP solution, pH-8.2-8.4. From this solution, 6.5-7 ul are pipetted on one end of a silanized slide (Sigma). The end of a 24x40 coverslip is placed at an angle, close to the DNA drop on the slide, so it touches the slide and then, in a quick move, that end of the coverslip is pressed against the slide with the thumb or a clamp. This makes the fluid spread between the slide and coverslip, from one end of the coverslip to the other. The flow of the solution is the driving force stretching the DNA. After 30-60 seconds the coverslip is removed. The slide is allowed to air -dry, and is placed for a minute each in jars with 70% and 100 % ethanol (fixation). The slide is covered with antifade/mounting medium containing 10-7 YOYO stain (Molecular Probes), then kept 3-5 minutes in the dark and examined at the microscope. Slide can be visualized and images taken using a regular green filter (for example FITC filter). Calculations indicated that, at least in our hands, the DNA is stretched to about 130% of its theoretical value, which is 34 uM for 1000bp (1kb). Details about the procedure will be further added after the publication of the submitted manuscript.
FISH on stretched DNA.
FISH on DNA fibers is useful in assessing the length of DNA probes, and to map probes relative to one another (Fig. 10j), as it can reveal even their degree of overlap. Thus, fiber FISH has superior mapping resolution compared to interphase FISH. There are many protocols for fiber-FISH or halo-FISH, and a discussion of all is beyond the purpose of this report, but several useful observations will be briefly mentioned. As a hybridization example, the figure below shows the extensive overlap between two PAC clones (red and green labeled, left side). On the right side, four cosmids (spanning a physical region of about 220kb) show a small overlapping area between the two clones (red and green) in the middle. A cosmid has an average size of about 35-40kb, whereas a PAC is usually 100-150 kb.
Why does hybridization resemble "beads on a string"?
If stretched DNA is stained with YOYO and observed through the FITC filter at the fluorescence microscope, immediately after exposure to light, the DNA fibers start breaking at many locations. From each break, the two free ends of DNA spring back to the nearest attachment point and coil, leaving a gap between them. These ends resemble little beads, and are thicker than the rest of the fiber. It is well known that when FISH is performed on stretched DNA/DNA fibers, hybridization always looks like an array of signals, or like "beads on a string" (Fig. 10j). The length of the array of dots is proportional to the physical length of the DNA probe. To determine precisely where the labeled DNA fragments bind on the DNA fibers, and where the "beads" are located, a simple experiment was done, in which a labeled probe was detected with a red fluorophore and the fibers were counterstained with a green fluorescent dye. We tested both YOYO (binds double-stranded DNA) and SYBR-Green II (binds single and double stranded DNA). In such experiments, performed with repeated or with single copy probes, we noticed that every red "dot" of the hybridized probe was also stained with green from either one of the counterstain dyes, but not every green dot was accompanied by a hybridization signal. The larger the hybridization signal the fainter the green color and vice-versa. When the DNA denatures, physical constraints within the double helix should allow some areas to become single stranded but at the expense of other regions, which should become overcoiled. Graphically, this would look similar to a chain of replication bubbles linked together. A double stranded DNA stain like YOYO should provide an image similar to beads on a string, but the fiber should be contiguous !!. Thus, SYBR-green II should show a contiguous signal for any DNA fiber. Experiments show that both dyes produce the same staining pattern, and that the DNA is broken during denaturing.
These observations suggest the following hybridization scenario in fiber FISH: the stretched DNA, when subjected to denaturing conditions (70%FA/2xSSC) at 75° to 95° C, actually breaks at many points. At each break, the two loose ends snap back towards the nearest attachment point and coil in a "dot-like" structure. The number and density of these dots depends on how many attachment points the initial DNA fiber has had to the slide, which, in turn, depends on the fixation procedure and the denaturing conditions. It is the DNA in the "dot-like" structures where denaturing takes place and where the labeled DNA probe binds. The stronger the hybridization, the more denaturing took place, and the less intense the YOYO signal. This explains easily the beads-on-a-string hybridization pattern.
Variability of signal-array length
Techniques for stretching genomic DNA, often expose the cellular material to detergents, chemicals or enzymes to release the DNA, immediately before stretching. The reason is the necessity to produce contiguous, long DNA fibers, important for FISH mapping. If fiber FISH is performed on such preparations, the arrays of signals obtained from the same probe may have different length. This is due to the variable degree of stretching of the genomic DNA, which may be only partial partially released from its chromatin structure. For a better assessment of the size of any probe, the longest array of hybridization signals should be the closest ot the real size.
Alternatively, genomic DNA can be freed from proteins through various treatment steps, while trapped in low melting point agarose blocks, which prevents the DNA fibers from breaking. The agarose is melted and DNA released right before use.
Tips for improving fiber-FISH hybridizations
- Fixation increases the number of attachment points between the DNA fiber and the slide surface. Chemical aging, 10-30 seconds at 94° C in 100% ethanol provides an easy and convenient way to fix the fibers. Alternatively, a simple incubation of the slides, for 1-2 minutes each, in 70% and 100% ethanol at room temperature may provide sufficient fixation. Baking the slides is also an alternative approach.
- Both simultaneous and separate denaturing protocols work for fiber FISH. Denaturing in a 70% FA/2xSSC solution works well, and higher temperatures (95° C) appear to denature the stretched DNA better than lower temperatures (70-75° C). A sudden chilling of the slide after the high denaturing temperature also improves the signals.
- For optimal FISH signals, the amount of labeled probe should be about twice or three times higher in fiber FISH compared with regular FISH.
- Blocking the repetitive sequences is also very important, otherwise hybridization signals will not be identifiable from all other background signals. Thus, a higher than usual (at least double) Cot1 amount should be used, compared to regular FISH.
- For the same reason, very stringent posthybridization washes should be used, to remove non-specifically bound, repetitive fragments. The charged slide surface on which the DNA is stretched may have higher affinity for some fluorescent dyes. To decrease the nonspecific binding of a dye or of an antibody to the slide surface, a 10 minute incubation of the slide in 0.1-1mg/ml BSA or other blocking reagent can be used.