Georges F. Carle

Orthogonal-field-alternation gel electrophoresis.

Publisher Summary This chapter discusses the protocols for separating large deoxyribonucleic acid (DNA) molecules by orthogonal-field alternation gel electrophoresis (OFAGE). The chapter discusses some of the practical and theoretical issues that affect the technique. The chapter also provides sufficient details to allow the assembly of an apparatus, which is functionally identical to one whose characteristics have been described previously. In 1983, when Schwartz et al. reported that the electrophoretic mobilities of DNA molecules up to at least several hundred kilobase pairs become strongly size dependent when they are electrophoresed in the presence of two alternately applied, approximately perpendicular electric fields. Use of this technique has allowed the analysis of many previously uncharacterized DNA molecules, such as the intact chromosomal DNA molecules of yeast and several protozoans. The chapter discusses the procedure for sample preparation and mentions that as the size of DNA molecules increases, it becomes progressively more difficult to keep them intact. Both mechanical shear and nucleolytic degradation must be controlled to produce acceptable OFAGE samples. To handle intact DNA molecules larger than 500 kb, however, it has proved necessary to prepare DNA samples by in situ lysis of cells or spheroplasts in a semisolid matrix. The two most largely used methods employ an agarose matrix, either as a solid plug or in the form of microbeads. The chapter concludes with few comments on apparatus design and some likely future directions of large-DNA electrophoresis. The most perplexing issue in this area concerns the field geometry. As discussed in the chapter, the electrophoretic results are extremely sensitive to this variable. Changes of even a few millimeters in the positioning or lengths of the electrodes—particularly the short electrodes have substantial effects.

7 – Orthogonal-Field-Alternation Gel Electrophoresis

Publisher Summary This chapter focuses on the detailed protocols for separating large DNA molecules by orthogonal-field-alternation gel electrophoresis (OFAGE). As the size of DNA molecules increases, it becomes progressively more difficult to keep them intact. To handle intact DNA molecules larger than 500 kb, it has proved necessary to prepare DNA samples by in situ lysis of cells or spheroplasts in a semisolid matrix. OFAGE results are influenced by an unusual number of interdependent variables. The functionally critical feature of an OFAGE apparatus is the positioning of the electrodes in relation to the gel and the perimeter of the electrophoresis chamber. OFAGE involves an element of brinksmanship in which the standard electrophoresis conditions border closely on disaster. It is probable that this situation is a direct reflection of the underlying fractionation principle, which is largely based on the retardation of large DNA molecules by interactions with the gel matrix that are only barely reversible.