Construction of Lambda Libraries from Large PFGE Fragments

Abstract

Pulsed-field gel electrophoresis (PFGE) has the capacity to fractionate large fragments of DNA up to thousands of kilobases in size. This aspect of the technique has been exploited for constructing long-range restriction maps of chromosomes from many different species including humans (see Chapters 14 , 15 , and 18 ). Besides its use for analytical purposes, PFGE has also been used as a preparative tool. Intact DNA obtained from preparative PFGE gels has been used for cloning into yeast artificial chromosome (MC) vectors (see Chapter 16 ) and for constructing jumping libraries (1). In addition, DNAeluted from PFGE gels has been used for generating libraries with a smaller insert size (2-7). In this latter procedure, DNA from a somatic cell hybrid is digested with a rare-cutting restriction enzyme, separated by PFGE, and the DNA from a particular PFGE fragment is eluted, digested, and cloned into a plasmid or phage vector. The resulting library is then screened with a species-specific probe to identify DNA segments from the donor chromosome of the hybrid. This use of preparative PFGE has had widespread application in the cloning of DNA close to several important disease genes, namely cystic fibrosis (4,6), Duchenne muscular dystrophy (2), choroideremia (7), polycystic kidney disease (3), and Huntington disease (5).