Construction of long-range restriction maps in human DNA using pulsed field gel electrophoresis

Abstract

Pulsed field gel electrophoresis (PFGE) is a powerful new tool for genetic analysis that can be applied to a variety of problems concerning genome structure and organization. This technique uses an agarose gel matrix to separate DNA molecules in a size range from 40 kb to 2,000 kb, molecules far larger than the maximum separable using standard agarose gel electrophoresis. The PFGE method can be used to separate the intact chromosomes from lower eukaryotes or to separate very large DNA fragments from higher eukaryotes generated by digestion with restriction endonucleases whose cleavage sites are rare. This paper describes the use of PFGE for construction of long-range restriction maps in the human genome and includes detailed methods for all steps. A pulsed field gel device that utilizes a rotating platform for altering the applied electric field is also described. Map construction is illustrated using a cloned DNA fragment (D3S2) from human chromosome 3. Several technical problems specific for mammalian genomes are discussed.