DHPLC Analysis

                        Regardless of the operation mode, the DNA samples are placed in the cooled 96-well autosampler compartment of the DHPLC system. It is controlled by the software from the manufacturer of the system (e.g. the WAVEMAKER software for the WAVE DNA Fragment Analysis System from Transgenomic). 

                         All the analytical parameters are entered into the program, including the sample information (sample identity and volume to be injected), application types, column temperature (mode of HPLC), and the linear gradient profile (the percentage of acetonitrile in the mobile phase). A linear gradient of 1.8–2.0% per  minute at a flow rate of 0.9 ml/min is usually used. In general, the start- and end-points of the gradient are adjusted according to the size of the DNA fragments. 

                          For the detection of unknown mutation, the optimal column temperature and the linear gradient profile are calculated by the algorithm of the software. The gradient profile for each injection includes the column regeneration and equilibrium steps prior to the next injection. The eluted DNA fragments are shown as peaks in the chromatogram. The retention time and the intensity of the peaks (height or area) are determined by the software.