Fluorescently marked DNA fragments electrophoretic mobility compensation in genetic analysis

The main factor of adjacent peaks uneven distribution in the experimental graphs during the nucleotide sequences determination which are recorded on four color channels outputs of genetic analyzer fluorescence detector is discussed in the article. The reason of this is the difference in the electrophoretic mobility of fluorescently marked DNA fragments. DNA fragments electrophoretic mobility differences compensation method is suggested and detailed sequence of calculations is given. The experimental data of plasmid DNA fragment separation is taken as an example. The peak time values and the unevenness of the original sequence of peaks in the four color channel fluorescence detector are determined, fluorescently marked DNA fragments electrophoretic mobility differences compensation results are estimated. Graphs of small segments of peaks sequence reflecting the results of the fragments separation at compensating differences in electrophoretic mobility are shown. The peak time shift parameters optimization for each color channel is made and refined graphs are built in the range of up to 300 nucleotides. It is also shown that the errors in determining the peaks time position are reduced significantly and have a random character. Horizontal axis calibration and linearization can be used in order to expand the proposed method applicability. The proposed method provides a reliable determination of missing and "extra" peaks.