28] Purification, specific fragmentation, and separation of large DNA molecules

Abstract

This chapter discusses the processes related to the purification, specific fragmentation, and separation of large deoxyribonucleic acid (DNA) molecules. The chapter mentions the development of three new techniques that allow the fractionation and analysis of DNA molecules on a size scale much larger than previously possible. The current methods permit the routine handling of DNAs up to 1.5 million base pairs (bp). The first technique involves the preparation of unbroken genomic DNAs inside agarose gels. The second involves the digestion of such DNAs in agarose with restriction nucleases that produce discrete, large fragments. The third technique, pulsed-field gel (PFG) electrophoresis, allows the size separation of DNAs ranging from 10,000 bp (10 kb) to more than 1.5 million bp (1.5 Mb). The chapter describes the new techniques and demonstrates examples of their applicability for the analysis of bacterial genomes and unicellular eukaryotic genomes. The chapter describes the procedure for preparing intact DNA. Ordinary DNA preparative procedures are carried out in solution. In a typical DNA preparation, the walls of the cells are first removed by appropriate enzymatic treatment. The resulting spheroblasts or protoplasts are then broken open by the destruction of their cell membranes with detergents and a metal chelator. This produces a complex mixture of DNA, ribonucleic acid (RNA), and proteins. Treatment of the mixture with proteases and RNases may remove some of the unwanted components. Additional proteins are removed by chemical extraction of the DNA solution with phenol, and the DNA is concentrated by alcohol precipitation and centrifugation. To obtain clean DNA preparations that do not have contaminants, which interfere with subsequent analytical or preparatory procedures, it is frequently necessary to repeat some of these steps. It is also essential that, during preparation, DNAs are not exposed unnecessarily to DNases. The chapter also discusses the pulsed-field gel electrophoretic separation of intact chromosomal DNA molecules, the specific fragmentation of high-molecular-weight DNA, and the applications of large DNA technology for the study of organisms with simple genomes.