Abstract
DNA sequencing by microchip capillary electrophoresis (CE) enables cheap, high-speed 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 σ2 < 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 power-modulated 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 σ2 ≈4 × 10-4, lower than required for the detection of single base-pair insertion or deletion in an optofluidic chip.
Highlights
A lab on a chip [1 3] squeezes the functionalities of a biological or chemical laboratory onto a single substrate
DNA sequencing by microchip capillary electrophoresis (CE) enables cheap, highspeed analysis of low reagent volumes
We demonstrate a variance of 2 4 10-4, lower than required for the detection of single base-pair insertion or deletion in an optofluidic chip
Summary
A lab on a chip [1 3] squeezes the functionalities of a biological or chemical laboratory onto a single substrate. The sorting and sizing of DNA molecules within the human genome project [7] has been enabled largely by CE separation and analysis [8] and has led to the genetic mapping of various human illnesses [9]. Application of CE-based DNA sequencing in a lab-on-a-chip to identify genomic deletions or insertions associated with genetic illnesses critically depends on the detection of single basepair insertions or deletions from DNA fragments in the diagnostically relevant range of 150 1000 base-pairs, i.e., it requires a variance of 2 < 10-3, while only 2 < 10-2 were reported [32, 6, 33]. We test different calibration strategies and demonstrate CE-based DNA analysis in an optofluidic chip with sub-base-pair resolution ( 2 4 10-4) of low concentrations of DNA molecules, thereby paving the way for the envisaged application
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