Purification and Isolation of Nucleic Acids

                         Conventionally, nucleic acids are analyzed by gel electrophoresis for the purposes of separation, identification, and purification. However, the process of the gel-based analysis involves labor-intensive steps such as sample and gel preparations, sample loading, gel staining, and photographic processing. 

                        The DHPLC system has high resolving power and thus allows the automatic purification and isolation of nucleic acids. It has been demonstrated that dsDNA, ssDNA, and RNA can be separated, quantified, and then recovered by the fragment collector of the DHPLC system. Under nondenaturing conditions, dsDNA molecules such as PCR products and restriction fragments are separated. The isolated dsDNA fragments can then be collected for downstream applications such as sequencing and cloning. In purification and isolation of ssDNA, DHPLC can directly separate ssDNA from dsDNA under fully denaturing conditions (75°C). 

                         The purification is facilitated by using a tagged primer, which has a hydrophobic moiety such as a biotin group or fluorescein, in PCR. The hydrophobicity of the ssDNA generated by the tagged primer is increased and leads to the increased retention time in DHPLC analysis. As a result, the two ssDNA species from the dsDNA PCR products can be separated. DHPLC is a simpler and faster method of purifying ssDNA than other techniques involving a variety of analytical molecular biology procedures. Moreover, the fully denaturing conditions can be applied to the purification and quantification of mRNA from total RNA. 

                           In DHPLC analysis, RNA degradation and spurious transcription can also be detected, and hence the quality and integrity can be determined. DHPLC greatly improves the analysis and purification of RNA as compared to the conventional methods by simplifying the lengthy experimental procedures.