Analytical methods oligonucleotides

A series of reviews has been published on the analysis of oligonucleotides using CE [183-185]. A comprehensive review on separation of mono, oligo- and polymeric nucleic acids by this method has also been published [186], In contrast to RP-HPLC and ion-exchange chromatography, CE - which has been established as the third technique of choice -is a purely analytical method [187]. Ion-exchange HPLC fails particularly with SODNs [185]. RP-HPLC does not at present allow baseline separation of SODNs, which differ by only one base pair [188]. Thus, control of preparative purifications by RP-HPLC has become an important application of CGE. Table 11 illustrates the resolution efficiency of CGE when compared with other methods. 

Table 11. Limits of resolution in detection of unmodified oligonucleotides using different analytical methods. Size limitations for baseline separation of ODNs differing by a single base-pair. Modified from [189],...

In addition to enzymes and other proteins, DNA and RNA have been successfully targeted with DCC systems in several studies. Oligonucleotide duplex formation is a highly efficient means to generate stable complexes even at very low concentration and these complexes can be easily studied by a range of different analytical methods. 

The resulting oligonucleotide is often of surprising purity as judged by analytic HPLC or electrophoresis, and up to 30 mg of a deoxyeicosanucleotide (20-base DNA) can be routinely obtained. Nevertheless small amounts of short sequences, resulting from capping and from base-catalysed hydrolysis, must always be removed by quick gel filtration, repeated ethanol precipitation from water (desalting), reverse-phase HPLC, gel electrophoresis, and other standard methods. 

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