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HEINZ-ULRICH G. WEIER
ing, the coverslips are removed, the slides are rinsed briefly with doubledistilled water, air dried and stored at 4°C.
The DNAs from plasmid, cosmid, Pl/PAC and BAC clones are isolated
using an alkaline lysis protocol and inserts are sized by PFGE. Digestion of
DNA with a rare cutting restriction enzyme produces linear high molecular weight DNA molecules, but the alkaline lysis procedure typically provides sufficient amounts of nicked circular or randomly broken DNA suitable for QDFM (Wang et aL 1996). In general, the DNA is loaded onto a
1.0% low melting point agarose gel and electrophoresed for about 15 h.
The band containing the desired linear or circular DNA is then excised
from the gel, and the gel slice is digested with agarase. Similarly, YAC
DNA from various clones is purified by PFGE. The integrity of DNA molecules can be assessed by microscopic inspection of aliquots of DNA
stained with 0.5 11M YOYO-1, before high molecular weight DNAs are
used for DNA fiber or FISH probe preparation or stored at 4°C in 100
mM NaCl.
The density of DNA molecules after DNA fiber stretching can be adjusted by altering the concentration of the DNA molecules prior to binding. Figure 2 shows the typical density of hybridized lambda DNA molecules. In experiments depositing circular PI and BAC DNA molecules, the
fraction of intact DNA molecules sometimes reaches -80%. While binding
ofDNA 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 (Fig. 3B). Mapping onto circular molecules
can thus be used for a 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 (Fig. 3B).
Quantitative DNA Fiber Mapping can facilitate the construction of
high resolution physical maps comprised of any combination of cosmid,
PI, PAC or BAC clones in two ways: if a low resolution map is available, for
example, in the form of a YAC contig, individual clones can be mapped
directly onto DNA fibers prepared from the larger clones (Weier et al.
1995, Cheng and Weier 1997). Alternatively, a high resolution map can
be constructed by measuring the extent and orientation of overlap between individual clones (Fig. 1). In most experiments, the applied scheme
will be determined by the sources of the clones and might combine both
schemes. Figure 3 shows some typical examples of QDFM applications.
Figure 3A shows the mapping of a PI clone (red) onto a colinear YAC
clone (green). Precise localization of the region of overlap is facilitated by
probes that mark specifically the ends of the YAC molecules (red).
HEINZ-ULRICH G. WEIER
ing, the coverslips are removed, the slides are rinsed briefly with doubledistilled water, air dried and stored at 4°C.
The DNAs from plasmid, cosmid, Pl/PAC and BAC clones are isolated
using an alkaline lysis protocol and inserts are sized by PFGE. Digestion of
DNA with a rare cutting restriction enzyme produces linear high molecular weight DNA molecules, but the alkaline lysis procedure typically provides sufficient amounts of nicked circular or randomly broken DNA suitable for QDFM (Wang et aL 1996). In general, the DNA is loaded onto a
1.0% low melting point agarose gel and electrophoresed for about 15 h.
The band containing the desired linear or circular DNA is then excised
from the gel, and the gel slice is digested with agarase. Similarly, YAC
DNA from various clones is purified by PFGE. The integrity of DNA molecules can be assessed by microscopic inspection of aliquots of DNA
stained with 0.5 11M YOYO-1, before high molecular weight DNAs are
used for DNA fiber or FISH probe preparation or stored at 4°C in 100
mM NaCl.
The density of DNA molecules after DNA fiber stretching can be adjusted by altering the concentration of the DNA molecules prior to binding. Figure 2 shows the typical density of hybridized lambda DNA molecules. In experiments depositing circular PI and BAC DNA molecules, the
fraction of intact DNA molecules sometimes reaches -80%. While binding
ofDNA 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 (Fig. 3B). Mapping onto circular molecules
can thus be used for a 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 (Fig. 3B).
Quantitative DNA Fiber Mapping can facilitate the construction of
high resolution physical maps comprised of any combination of cosmid,
PI, PAC or BAC clones in two ways: if a low resolution map is available, for
example, in the form of a YAC contig, individual clones can be mapped
directly onto DNA fibers prepared from the larger clones (Weier et al.
1995, Cheng and Weier 1997). Alternatively, a high resolution map can
be constructed by measuring the extent and orientation of overlap between individual clones (Fig. 1). In most experiments, the applied scheme
will be determined by the sources of the clones and might combine both
schemes. Figure 3 shows some typical examples of QDFM applications.
Figure 3A shows the mapping of a PI clone (red) onto a colinear YAC
clone (green). Precise localization of the region of overlap is facilitated by
probes that mark specifically the ends of the YAC molecules (red).