Direct DNA quantification in cell lysates by fluorometric method in combination with an advanced chemometric model

Abstract

Accurate quantification of genomic DNA is fundamental to many molecular analyses such as diagnostic assays and genotyping. Due to relatively low and varying extraction rates of DNA from cell lysates by genomic DNA extraction kits, it is desired to quantitatively determine DNA contents directly in cell lysates. However, the coexistence of scatterers (e.g., cell debris), absorbers, and intrinsic fluorophores in cell lysates prevents either UV–Vis spectrophotometric methods or fluorometric methods based on fluorescent dyes and ratiometric models to be directly applied to cell lysates. In this contribution, a novel fluorometric method based on an advanced chemometric model which can effectively eliminate the effects of scatterers and absorbers on fluorescence spectra of cell lysates was proposed for the direct quantification of DNA in lysates of CEM cells. Without cumbersome prior DNA extraction, the proposed method achieved quite stable and accurate DNA quantification with recovery rates in the range of 89%–115%, considerably better than that of either the fluorometric method based on the ratiometric model or the UV–Vis spectrophotometric method coupled with DNA extraction. The proposed method has the advantages of rapidness, simplicity, and high precision, and can be developed into a promising alternative for accurate quantification of genomic DNA.