Quantification of Plasmid DNA in pCA-HA/poly-L-lysine Complex

Abstract

This report describes the comparison of four pDNA quantitative methods including UV/Visible spectro-photometry, agarose gel electrophoresis, EtBr staining fluoro-photometry and HPLCSEC method. Finally, the HPLC-SEC method was used to evaluate the interaction of plasmid DNA (pCA-HA) and poly-L-lysine (PLL). In the pCA-HA concentrations ranged form 3.125 to 50 μg/mL, the standard curve of pCA-HA has good linearity, accuracy (CV%＜3%) and precision (Error%＜4%) by using UV/Visible spectro-photometry. But the sensitivity is not enough to evaluate less than 3 μg/mL of pDNA. The intensity of fluorescene emission of EtBr stained pCA-HA is not dose dependent by using EtBr staining fluoro-photometry. The agarose gel electrophoresis had a high sensitivity (ng/mL level), but the accuracy (CV%＞15%) and precision (Error%＞15%) were not good in the range of 0.048~0.760 μg/mL pCA-HA. The standard curve of pCA-HA had good linearity, accuracy (CV%＜3%) and precision (Error%＜5%) in the pCA-HA concentration ranges from 0.4 to 1.2 μg/mL by using the HPLC-SEC method. PLL, a positive charged peptide, was used to condense DNA and enhance the transfection of DNA in cell. We used the HPLC-SEC method to evaluate the interaction of pDNA and PLL. When the charge of pCA-HA and PLL were almost the same, the peak of pDNA disappeared from the HPLC chromatogram. It means that the charge ratio of pCA-HA/PLL is close to 1 in the complex.