13] The isolation of extrachromosomal DNA by hydroxyapatite chromatography

Abstract

Publisher Summary This chapter discusses an experimental study focusing on the isolation of extrachromosomal deoxyribonucleic acid (DNA) by hydroxyapatite (HAP) chromatography. HAP has been widely used in the preparation and analysis of nucleic acids; it has a higher affinity for nucleic acid molecules with rigid and ordered structures than for those having disordered and flexible structures. In the study discussed in the chapter, which involved the analysis of DNA–DNA and DNA–RNA hybridization mixtures on HAP columns, it was consistently observed that high-molecular-weight native DNA molecules eluted very poorly in 0.4–0.5 M phosphate buffer (PB), pH 6.8, which is commonly used to elute the reannealed double-stranded DNA from HAP. This led to the development of a rapid and reproducible method for purifying the low-molecular-weight extrachromosomal DNA from eukaryotic cells virtually free of protein, RNA, and high-molecular-weight bulk cellular DNA. The purification of extrachromosomal DNA and the characterization of extrachromosomal DNA are also discussed in the chapter.