6 - Purification, Specific Fragmentation, and Separation of Large DNA Molecules

Abstract

This chapter discusses the purification, specific fragmentation, and separation of large DNA molecules. Three new techniques have been recently developed that allow the fractionation and analysis of DNA molecules on a size scale much larger than previously possible. Electrophoresis techniques developed in the past 50 years have allowed effective separation of proteins and nucleic acids on the basis of molecular weight. DNA molecules above 20 kb long usually cannot be separated by gel electrophoresis because they are larger than the pore size of the matrix. In standard gel electrophoresis, the electrical field is applied constantly in one direction. Pulse times used with PFG electrophoresis can be tuned such that maximal separation of particular sized molecules is obtained.