7] High-resolution nucleic acid separations by high-performance liquid chromatography

Abstract

A comparison of high-performance liquid chromatography (HPLC), with traditional electrophoretic and ultracentrifugal techniques, reveals that the weakest aspects of the traditional techniques are actually the strengths of HPLC. Automation of HPLC sampling, data reduction, fraction collection, and reporting functions enhances quantification and promotes greater throughput and recovery. HPLC also offers the opportunity for scaleup. In comparison, gel electrophoretic and ultracentrifugal methods are difficult to automate and quantify the present problems, with recovery, are not readily scaleable, and for some applications offer little practical advantage over current HPLC techniques (e.g., synthetic oligonucleotide separation). This chapter discusses the sections on applications and procedures for a wide variety of nucleic acids. Single-stranded (ss) nucleic acid separations include: (1) synthetic oligonucleotides (e.g., polymerase chain reaction (PCR) and sequencing primers, oligonucleotide probes, and ribozymes) and (2) phosphorothioate (antisense) oligonucleotides. Double-stranded (ds) nucleic acid separations include: (1) restriction fragments (RFs), (2) PCR products, and (3) plasmids.