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. 2019 Mar 16:17:100083.
doi: 10.1016/j.bdq.2019.100083. eCollection 2019 Mar.

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

Martin Hussels  1 , Susanne Engel  1 , Nicole Bock  1
Affiliations

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

Martin Hussels et al. Biomol Detect Quantif. .

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.

Keywords: DNA-copy concentration; Enumeration based quantification; Flow cytometric counting; Metrology; Molecular quantification; dPCR.

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Figures

Fig. 1
Fig. 1
Optical setup for detection of single stained DNA fragments. The laser beam is expanded by a telescope consisting of two spherical lenses and then shaped to an ellipse by a second telescope consisting of two cylindrical lenses. The shaped beam is focused into the flow channel of a conventional flow cell where it excites the stained DNA fragments. The fluorescence signal is collected in sideward direction by an aspheric lens and focused on an iris aperture acting as spatial filter. The light passing the spatial filter is focused on a single photon counting avalanche photo diode (APD) for detection. For spectral selection a 500 nm longpass filter and a 525 nm bandpass filter with 50 nm bandwidth are inserted in the detection pathway.
Fig. 2
Fig. 2
Comparison of peak area histograms of single measurements of dye containing dilution buffer (red) and diluted pUC19TB sample (black). Peak detection for both samples was applied using same settings to get comparable results. The background signal coming from the dilution buffer shows to be stable for both measurements making background subtraction feasible. This holds also for several repeat measurements not shown here.
Fig. 3
Fig. 3
Calibration of the fluorescence intensity of the DNA fragments over fragment length in base pairs (bp). Part a shows the histograms of the distribution of intensities measured for the different samples. Part b shows the resulting calibration curve. The data points and error bars in the graph are derived from Gaussian fits of the histograms in part a, whereby the error bars represent the standard deviation of the fitted gaussians. Linear regression of the data (red line) shows excellent linear relationship of fragment length and fluorescence intensity. The corresponding fragment sizes are 4316 bp, 8519 bp, 17038 bp, and 48502 bp.
Fig. 4
Fig. 4
Typical pulse shapes measured for pUC19TB plasmids (Raw data after baseline correction). A single plasmid event is shown in a and coincident detection of two plasmids resulting in a double or broader peak is shown in b and d. Part c shows either a plasmid dimer or perfectly coincident detection of two plasmids, which cannot be distinguished.
Fig. 5
Fig. 5
Density plot of peak area versus peak height of the pUC19TB sample. Three gates are shown, which are used to classify events as single plasmids (green), coincidences of two plasmids (orange), and potential dimers (blue).
See this image and copyright information in PMC

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