Chapter 4 High Performance Liquid Chromatography of Nucleic Acids

Abstract

Publisher Summary Isolation of altogether different but well-defined nucleic acids is of major interest in present-day biochemistry, biophysics, molecular biology and gene technology. For example, in biophysical chemistry of nucleic acids large amounts of homogeneous nucleic acid material are needed. The requirements range from synthetic oligonucleotides with defined sequences which are used to determine the elementary parameters of nucleic acid structure and stability, up to large natural nucleic acids such as plasmids which are the subject of studies about supercoiled structures and nucleic acid topology. For biochemical studies on RNA, e.g. tRNA, viroid RNA or viral RNA, highly purified fractions of these RNAs from natural sources have to be prepared. Often, one is confronted with the difficulty of those RNAs being present in the cell together with a vast excess of other, sometimes quite similar, nucleic acids. Other new applications of HPLC have emerged from recent developments in molecular biology and gene technology. Routinely, for the different steps of the cloning and sequencing procedures synthetic oligonucleotides, DNA fragments and plasmids must be prepared in great purity. Purity alone, however, does not suffice as a specification; in addition, they are supposed to be highly active in a variety of enzymatic reactions and biological transformations. Another new field of HPLC of nucleic acids arises in diagnostic applications for medicine and phytopathology. In these new areas of application too, highly purified probes of recombinant DNA or RNA will be required, e.g. for testing for infectious diseases or for genetically determined disorders.