1. Unless stated otherwise, all solutions should be prepared with purified water having a restistivity of 18.2 MQ-cm and total organic content of less than five parts per billion. This standard is referred to as "water" in this protocol.

2. Unless noted otherwise, all chemicals were purchased from Sigma.

3. The AL solution set (solutions I-III) is for isolation of P1, PAC, or BAC DNA.

4. Allow the antifade solution to warm up for only a short period, remove an aliquot, and then place it back into the freezer. Fresh antifade solution is colorless. Discard aliquot when solution has turned dark.

5. DNA isolated from P1, PAC, or BAC clones can be loaded directly into the wells of the PFGE gel. Use 1 ^L of loading buffer per 5 ^L of DNA solution.

6. The integrity of DNA molecules can be assessed by microscopic inspection of ali-quots of DNA stained with 0.5 ^M YOYO-1, before high molecular weight DNAs are used for DNA fiber preparation or stored at 4°C in 100 mM NaCl.

7. Slides from different manufacturers or the same brand but different batches may produce different qualities of DNA fibers. Purchase a sufficiently large batch of slides from one manufacturer such as Erie Scientific (Portsmouth, NH), a supplier of glass slides that are sold under Fisher or BD labels. Avoid slides that are painted on one end, since the paint might dissolve during pretreatment. Slides that have a sandblasted or etched area at one end are preferable.

8. Cover slip silanation is performed like the procedure described for slides. Briefly, cover slips are rinsed with distilled water and dehydrated in 100% ethanol. Cover slips are derivatized with a 0.05 to 0.1% solution of APS in 95% ethanol for 2 min. Cover slips are then rinsed and dried as described in Subheading 3.2.

9. Binding of DNA molecule ends to the substrate and the stretching effect can be monitored in the fluorescence microscope by staining the DNA with 0.2 to 0.5 yM YOYO-1 prior to deposition. This allows exclusion of batches of slides that bind DNA too tightly. Dilute the YOYO-1 in water, since the antifade solution prevents the DNA molecules from binding.

10. The density of DNA molecules after DNA fiber stretching can be adjusted by altering the concentration of the DNA molecules prior to binding. Figure 2D to F shows the typical density of hybridized X DNA molecules. In experiments depositing X, P1, PAC, or BAC DNA molecules, the fraction of intact DNA molecules can reach approx 50 to 70%. Mapping can utilize both linear and circular DNA molecules. Although binding of DNA molecules in their circular form helps to maintain their integrity, it interferes with DNA fiber stretching, and the molecules are found to be stretched to varying degrees. Mapping onto circular molecules can thus be used for rough estimation of overlap and mapping on linear fibers for high-precision measurements. This can be done in a single experiment, because some circular DNA molecules are sheared during deposition, thus providing randomly broken linear DNA molecules. (see ref. 53 for a more detailed description).

11. Different procedures have been described to stretch DNA molecules (47-51). In our hands, stretching involving a hydrodynamic force (meniscus) at 20°C or 4°C has proved most reproducible. There is, however, no need to wait until the preparation has dried to completion. Once the DNA molecules have bound to the substrate, the cover slip can be lifted to exert the hydrodynamic stretching force (53).

12. We found that even short periods of drying out of slides or parts of them during the immunocytochemical signal amplification lead to unacceptable levels of background fluorescence owing to nonspecific binding of detection reagents. It is important to just drain the liquids from the slides and then rapidly apply the next solution, a blocking solution, or antibodies.

13. If only two labels are used, i.e., biotin and digoxigenin, bound probes are detected with avidin-FITC DCS (Vector) and antidigoxigenin-rhodamine, respectively.

14. Most fluorochromes fade quickly. Under the microscope, we always minimize the exposure of slides to the excitation light. The key factor in producing good images is to use the DNA counterstain or one fiber probe to quickly localize areas showing a sufficiently high density of well-stretched molecules and then switch to the image acquisition mode.

15. Always measure additional segments of the molecule such as the vector segment since these might provide additional information about the extent and homogeneity of DNA stretching.

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