Combined microfluidic-optical DNA analysis with single-base-pair sizing capability

Abstract

DNA sequencing by microchip capillary electrophoresis (CE) enables cheap, highspeed analysis of low reagent volumes. One of its potential applications is the identification of genomic deletions or insertions associated with genetic illnesses. Detecting single base-pair insertions or deletions from DNA fragments in the diagnostically relevant size range of 150−1000 base-pairs requires a variance of σ 2 < 10 −3 . In a microfluidic chip post-processed by femtosecond-laser writing of an optical waveguide we CE-separated 12 blue-labeled and 23 red-labeled DNA fragments in size. Each set was excited by either of two lasers powermodulated at different frequencies, their fluorescence detected by a photomultiplier, and blue and red signals distinguished by Fourier analysis. We tested different calibration strategies. Choice of the fluorescent label as well as the applied fit function strongly influence the obtained variance, whereas fluctuations between two consecutive experiments are less detrimental in a laboratory environment. We demonstrate a variance of σ 2 ≈4 × 10 −4 , lower than required for the detection of single base-pair insertion or deletion in an optofluidic chip. 895-897 (1993). 5. G. J. M. Bruin, "Recent developments in electrokinetically driven analysis on microfabricated devices,"

Roadmap for optofluidics

“…Most importantly, the dependence of migration time on base-pair size needs to be understood theoretically. The results displayed in figure 25prove that a quadratic fit reproduces the measured data significantly better than a linear fit, indicating that the assumption of a simple logarithmic dependence of migration time on base-pair size underlying the production of DNA ladders with a logarithmic increase in base-pair size is not justified by the experiment [182]. Nevertheless, the sizing accuracy crucially depends on the applied law to fit the reference data.…”

“…The dependence of sizing accuracy on the important system parameters must be investigated more systematically and its theoretical background needs to be improved. For example, recently it has been identified that the choice of fluorescent dye label can significantly influence the sizing accuracy and that a proper choice can largely improve the accuracy [182]; see figure 25, but the reason for this effect is unknown and systematic investigations are lacking. In the same work it has been demonstrated that the calibration strategy influences the sizing accuracy [182].…”

“…For example, recently it has been identified that the choice of fluorescent dye label can significantly influence the sizing accuracy and that a proper choice can largely improve the accuracy [182]; see figure 25, but the reason for this effect is unknown and systematic investigations are lacking. In the same work it has been demonstrated that the calibration strategy influences the sizing accuracy [182]. On the one hand, it may not matter much whether the optofluidic device is calibrated by flow of a known reference sample in the same or in a separate experiment, and whether the DNA fragments of the reference sample are almost identical or slightly different from those of the unknown sample to be investigated.…”

“…Choice of a suitable dye label, combined with reference calibration and sample investigation by fluorescent detection in consecutive experiments, and application of a best-guess fit function, has resulted in capillary electrophoretic separation of fluorescent-labeled DNA molecules in the 150−1000 basepair range with single-base-pair resolution [182] (see figure 25), thereby paving the way towards the detection of single-base-pair insertion or deletion in a lab-on-a-chip with low reagent volumes in a few-minute experiment.…”