Column liquid chromatography has been employed for the separation, purification, and detection of nucleic acids. Among chromatography methods provided, anion-exchange chromatography has been most commonly employed for the isolation and purification of not only oligonucleotides but also large double-stranded DNA.
In anion-exchange chromatography, DNA retention is dependent on electrostatic interactions between the negatively charged phosphate groups of DNA and cationic sites of the chromatographic matrix (see the picture below).
DNA can be eluted from the anion-exchange column by altering the ionic strength of the buffer solution. In the presence of a buffer of increasing ionic strength, DNA retention is generally a function of the number of negative charges associated with the phosphate groups.
One of the most common anion-exchange materials is diethylamino ethyl (DEAE)-bonded support, and it has been extensively used to modify original soft-gel supports, porous microparticular silica, and polymer-based materials. Availability of porous microparticular silica and polymer-based supports has led to enhanced column resolution and faster analysis times. However, the columns packed with porous particles have been used only with limited success in the analysis of large double-stranded DNA fragments.
HPLC columns based on nonporous, small-particle resins are available, it is very fast and also efficient in separating large double-stranded DNA. The success of these columns is based on the fact that nonporous, small particle size material with which they are packed provides improved efficiency and short analysis time. This is the result of a much faster DNA transfer between the solid and liquid column phases than is possible for conventional porous support. In addition, these columns offer high DNA recoveries because of the absence of pores.
HPLC of nucleic acids and PCR products has been successfully employed for post-PCR analyses.