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Journal of Histochemistry and Cytochemistry, Vol. 49, 939-948, August 2001, Copyright © 2001, The Histochemical Society, Inc.

REVIEW

DNA Fiber Mapping Techniques for the Assembly of High-resolution Physical Maps

Heinz-Ulrich G. Weiera
a Department of Subcellular Structure, Life Sciences Division, University of California, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, California

Correspondence to: Heinz-Ulrich G. Weier, Dept. of Subcellular Structure, Life Sciences Division, MS 74-157, U. of California, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720. E-mail: ulliweier@hotmail.com

High-resolution physical maps are indispensable for directed sequencing projects or the finishing stages of shotgun sequencing projects. These maps are also critical for the positional cloning of disease genes and genetic elements that regulate gene expression. Typically, physical maps are based on ordered sets of large insert DNA clones from cosmid, P1/PAC/BAC, or yeast artificial chromosome (YAC) libraries. Recent technical developments provide detailed information about overlaps or gaps between clones and precisely locate the position of sequence tagged sites or expressed sequences, and thus support efforts to determine the complete sequence of the human genome and model organisms. Assembly of physical maps is greatly facilitated by hybridization of non-isotopically labeled DNA probes onto DNA molecules that were released from interphase cell nuclei or recombinant DNA clones, stretched to some extent and then immobilized on a solid support. The bound DNA, collectively called "DNA fibers," may consist of single DNA molecules in some experiments or bundles of chromatin fibers in others. Once released from the interphase nuclei, the DNA fibers become more accessible to probes and detection reagents. Hybridization efficiency is therefore increased, allowing the detection of DNA targets as small as a few hundred base pairs. This review summarizes different approaches to DNA fiber mapping and discusses the detection sensitivity and mapping accuracy as well as recent achievements in mapping expressed sequence tags and DNA replication sites.

(J Histochem Cytochem 49:939–948, 2001)

Key Words: physical mapping, DNA fibers, hybridization, FISH, digital image analysis


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