Chapter 18
PROTOCOL
Quantitative DNA Fiber Mapping
HEINZ-ULRICH G. WEIER
Introduction
High resolution physical maps have become indispensable for the positional cloning of disease genes and large scale sequencing projects. Common maps are based on ordered sets of clones from sources such as cosmid, PI/PAC/ BAC, or yeast artificial chromosome (YAC) libraries. The
assembly of such maps is facilitated by application of fluorescence in
situ hybridization (FISH). Hybridization of non-isotopically labeled
probes onto preparations of DNA molecules ('DNA fibers') that were
bound with one or both ends to a solid substrate and stretched homogeneously, forms the base of our 'Quantitative DNA Fiber Mapping (QDFM)'
technique. Because the DNA fibers are easily accessible to probes and detection reagents, hybridization efficiencies are typically high and allow
DNA targets as small as 500-1000 bp to be detected routinely. Quantitative
DNA fiber mapping experiments require only standard laboratory equipment and access to a fluorescence microscope. By hybridizing one clone
onto another, the extent and orientation of overlaps can be quantitated
with near kilobase resolution. To measure the physical distance between
non-overlapping DNA fragments, probes are hybridized to DNA fibers
representing a larger genomic interval (Fig. 1). This also allows the mapping of expressed sequences (cDNAs) along DNA fibers representing
genomic DNA.
Heinz-Ulrich G. Weier, University of California, E.O. Lawrence Berkeley National
Laboratory, Department of Subcellular Structure, Life Sciences Division, M.S. 74-157,
1 Cyclotron Road, Berkeley, CA, 94720, USA (phone +510-486-5347; fax +510-4865343; e-mail ugweier@lbl.gov)
PROTOCOL
Quantitative DNA Fiber Mapping
HEINZ-ULRICH G. WEIER
Introduction
High resolution physical maps have become indispensable for the positional cloning of disease genes and large scale sequencing projects. Common maps are based on ordered sets of clones from sources such as cosmid, PI/PAC/ BAC, or yeast artificial chromosome (YAC) libraries. The
assembly of such maps is facilitated by application of fluorescence in
situ hybridization (FISH). Hybridization of non-isotopically labeled
probes onto preparations of DNA molecules ('DNA fibers') that were
bound with one or both ends to a solid substrate and stretched homogeneously, forms the base of our 'Quantitative DNA Fiber Mapping (QDFM)'
technique. Because the DNA fibers are easily accessible to probes and detection reagents, hybridization efficiencies are typically high and allow
DNA targets as small as 500-1000 bp to be detected routinely. Quantitative
DNA fiber mapping experiments require only standard laboratory equipment and access to a fluorescence microscope. By hybridizing one clone
onto another, the extent and orientation of overlaps can be quantitated
with near kilobase resolution. To measure the physical distance between
non-overlapping DNA fragments, probes are hybridized to DNA fibers
representing a larger genomic interval (Fig. 1). This also allows the mapping of expressed sequences (cDNAs) along DNA fibers representing
genomic DNA.
Heinz-Ulrich G. Weier, University of California, E.O. Lawrence Berkeley National
Laboratory, Department of Subcellular Structure, Life Sciences Division, M.S. 74-157,
1 Cyclotron Road, Berkeley, CA, 94720, USA (phone +510-486-5347; fax +510-4865343; e-mail ugweier@lbl.gov)