Investigation of direct counting and sizing of DNA fragments in flow applying an improved data analysis and correction method

Abstract

Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Additionally, comparison of this method to the orthogonal method of digital PCR is useful with the restriction of low specificity of the direct detection method. In this study, we analysed raw detector signals and the sizing performance for target identification and the effect of coincidence detection concerning concentration measurements. We present data of purified artificial DNA samples measured with the home-built setup. Main emphasis was to develop an improved data analysis method to gain insight into and carefully correct for coincident detection of DNA fragments and for estimation of the amount of fragment dimers.