FiberFISH - Fig 1 Fig. 1. Microscopic measurements of DNA fibers All microscopic images (including the ruler in panel G) were captured with the 100x objective and are shown at the same scale. Panel A shows several lambda DNA fibers, whereas panel C shows a summary of the most frequent geometric forms taken by the DNA fibers on a slide. For comparison purposes, the gel image shown in panel B (restriction digest) includes the same three BACs depicted as DNA fibers in figures D, E and F. Figures I, J, K and L show DNA fibers of various sizes (cosmid, plasmid, BAC, PAC). With the exception of panel B, all numbers indicate the length of DNA fibers in pixels. The multiple breaks seen in some fibers were induced by light energy, during image visualization and capturing. In assessing DNA length, please note that all DNA probes measured include the respective vector DNA. (A) lambda phage DNA (48.5kb) used as a control, with a measured size of 158-159 pixels. (B) BamHI digestion of three different, but partially overlapping PACs, on agarose gel stained with ethidium bromide. Based on gel analysis, PAC no. 1 was 137kb, PAC no. 2 was 139 kb and PAC no. 3 was 91kb. M = marker (1kb ladder (GIBCO) + lambda-HindIII + uncut lambda DNA). Numbers close to small lines indicate fragment sizes (kb), whereas numbers without lines indicate marker sizes. Compare with D-F. (C) Examples of fiber shapes seen when stretching any circular DNA molecule (P1 DNA shown). Closed circles (251, 257, 271) have comparable lengths, whereas collapsed circles (138, 153) have shorter, thicker fibers. Bends in the fibers indicate less pulling force on the DNA and result in less stretching of the molecule. Five fully knotted DNA molecules are also shown.(D) PAC no. 1 fibers (see also 1B). The sizes of the circular and collapsed circle DNAs of PAC no. 1 correspond to a 135 kb molecule, similar to the restriction digest value(s). The longest linear molecule found on this slide (346 pixels) was shorter than expected, indicating that the DNA was mostly sheared during isolation. The 182- and 190- pixel DNAs are collapsed circles folded back onto themselves (not usable for sizing). (E) PAC no. 3 fibers (see also 1B). The closed circle (260) is longer than the twisted circle (200), indicating a higher degree of DNA stretching. A nick in the DNA molecule of the collapsed circle (137) shows clearly that in this geometrical figure, the DNA molecule folds back onto itself. Size measurements correspond to a 105 kb DNA molecule. (F) PAC no. 2 fibers (see also 1B). The sizes of the collapsed circle (182) and the linear fiber (441) correspond to a DNA of approximately 138 kb. The small knotted areas (arrows) make the closed circles appear somewhat shorter than expected. (G) Microscopic ruler photographed through the 100x objective. The double-headed arrow corresponds to 10 µm. (H) Fiber-FISH of a BAC probe labeled with Cy3 and hybridized onto genomic DNA fibers, obtained by SDS-incubation of cells directly on slides. Because the SDS-protocol stretches genomic DNA fibers to different degrees, the same BAC probe yields arrays of hybridization signals varying tremendously in length.(I) Cosmid DNA fibers, corresponding to approximately 38-40 kb. (J) Linear fibers of an 8.6kb plasmid (vector + insert). (K) The twisted, circular molecule of a sequenced BAC clone (211.3 kb). The 555 pixels correspond to a DNA molecule of approximately 222 kb (insert + vector), which is close to the real value. (L) DNA fibers of a 95kb PAC clone, showing numerous breakages induced by light, during image capturing at the microscope.
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