18 Quantitative DNA Fiber Mapping
249
found on the same slides are stretched more homogeneously, thus providing DNA fibers without need for normalization.
Expressed sequences can be mapped easily by QDFM, if each individual target extends for a few hundred base pairs or more. A common approach hybridizes small genomic DNA fragments of 1-2 kb that contain
known exons onto larger genomic DNA molecules. If the eDNA sequence
and some information about intron-exon boundaries are available, such
small DNA fragments can rapidly be generated from genomic DNA using
PCR. Figure 3C demonstrates this by mapping exon 2 of the human Band
4.1 gene onto a homologous BAC molecule. This allowed the localization
of the -2 kb exon with near kilobase precision.
Figures 3D,E illustrate the application of QDFM for measurement of
BAC clone overlap. In such experiments, the DNA fiber is counterstained
by a probe detected in blue. A BAC vector-specific probe (green) and a
PCR-generated probe of -1300 bp (red) that binds close to the T7 end
of the vector are included to highlight the vector part of the DNA fibers
(Figs. 3C,D). The two BAC clones shown in Fig. 3D overlap by approximately 80 kb and the overlapping region is close to the SP6 promoter in
the BAC vector (Fig. 3D,E). These results are summarized schematically in
Fig.3E.
Arapid approach to studying the genomic organization ofgenes relies on
direct mapping of expressed sequences. The probe DNA is isolated from
cDNA clones, labeled and hybridized onto genomic DNA fibers. In the
presence of blocking DNA, the cDNA probes will bind specifically to their
complementary DNA targets, i.e., exons and 5- or 3-untranslated regions
(UTRs) along the DNA fiber. This leaves non-coding regions (introns, 5- and
3-flanking DNA) unstained (Fig. 3F). Using FISH conditions similar to those
..
Fig. 3. Quantitative DNA Fiber Mapping (QDFM) using large insert human genomic DNA
clones. A Mapping PI clones along YAC molecules. The arrows indicate the distance from
the respective ends. B Circular DNA molecules excised from a PFGE gel purified and stained
with YOYO-I revealed closed circular DNA molecules in the presence of linear molecules of
different length. C Physical mapping ofsmall cDNA clones in larger genomic intervals. DNA
fibers (blue) prepared from a BAC clone were hybridized with a -2 kb insert of a plasmid
containing exon 2 of the human Band 4.1 gene (red). D, E Determination of overlap between
linked BAC clones. A probe prepared from BAC # 103 (red) was hybridized onto DNA fibers
prepared from BAC #97 (blue). E shows a schematic representation. D Mapping the genomic
organization of expressed sequences. Here, BAC DNA fibers (blue) were hybridized with a
-5 kb cDNA probe (red). Three hybridization domains representing larger exons and the 3
UTR were detected on the DNA fibers. The green probe in C, D and F delineates the BAC
vector
249
found on the same slides are stretched more homogeneously, thus providing DNA fibers without need for normalization.
Expressed sequences can be mapped easily by QDFM, if each individual target extends for a few hundred base pairs or more. A common approach hybridizes small genomic DNA fragments of 1-2 kb that contain
known exons onto larger genomic DNA molecules. If the eDNA sequence
and some information about intron-exon boundaries are available, such
small DNA fragments can rapidly be generated from genomic DNA using
PCR. Figure 3C demonstrates this by mapping exon 2 of the human Band
4.1 gene onto a homologous BAC molecule. This allowed the localization
of the -2 kb exon with near kilobase precision.
Figures 3D,E illustrate the application of QDFM for measurement of
BAC clone overlap. In such experiments, the DNA fiber is counterstained
by a probe detected in blue. A BAC vector-specific probe (green) and a
PCR-generated probe of -1300 bp (red) that binds close to the T7 end
of the vector are included to highlight the vector part of the DNA fibers
(Figs. 3C,D). The two BAC clones shown in Fig. 3D overlap by approximately 80 kb and the overlapping region is close to the SP6 promoter in
the BAC vector (Fig. 3D,E). These results are summarized schematically in
Fig.3E.
Arapid approach to studying the genomic organization ofgenes relies on
direct mapping of expressed sequences. The probe DNA is isolated from
cDNA clones, labeled and hybridized onto genomic DNA fibers. In the
presence of blocking DNA, the cDNA probes will bind specifically to their
complementary DNA targets, i.e., exons and 5- or 3-untranslated regions
(UTRs) along the DNA fiber. This leaves non-coding regions (introns, 5- and
3-flanking DNA) unstained (Fig. 3F). Using FISH conditions similar to those
..
Fig. 3. Quantitative DNA Fiber Mapping (QDFM) using large insert human genomic DNA
clones. A Mapping PI clones along YAC molecules. The arrows indicate the distance from
the respective ends. B Circular DNA molecules excised from a PFGE gel purified and stained
with YOYO-I revealed closed circular DNA molecules in the presence of linear molecules of
different length. C Physical mapping ofsmall cDNA clones in larger genomic intervals. DNA
fibers (blue) prepared from a BAC clone were hybridized with a -2 kb insert of a plasmid
containing exon 2 of the human Band 4.1 gene (red). D, E Determination of overlap between
linked BAC clones. A probe prepared from BAC # 103 (red) was hybridized onto DNA fibers
prepared from BAC #97 (blue). E shows a schematic representation. D Mapping the genomic
organization of expressed sequences. Here, BAC DNA fibers (blue) were hybridized with a
-5 kb cDNA probe (red). Three hybridization domains representing larger exons and the 3
UTR were detected on the DNA fibers. The green probe in C, D and F delineates the BAC
vector