Abstract
We demonstrate the use of open-surface microfluidics to sequence DNA by pyrosequencing at the plain hydrophobically coated surface of a microscope glass cover slip. This method offers significant advantages in terms of instrument size, simplicity, disposability, and functional integration, particularly when combined with the broad and flexible capabilities of open-surface microfluidics. The DNA was incubated on superparamagnetic particles and placed on a hydrophobically coated glass substrate. The particles with bound DNA were moved using magnetic force through microliter-sized droplets covered with mineral oil to prevent water evaporation from the droplets. These droplets served as reaction "stations" performing pyrosequencing as well as washing stations. The resequencing protocol with 34-mer single-stranded DNA (ssDNA) was used to determine the reaction performance. The de novo sequencing was performed with 51-mer and 81-mer ssDNA. The method can be integrated with previously shown sample preparation and PCR into a single sample-to-answer system on a plain glass surface.
Highlights
Deoxyribonucleic acid (DNA) molecules carry the majority of genetic information for all living species as well as viruses
Performing the pyrosequencing reactions without the washing step resulted in a 30% carry-over signal by the DNA/primer/superparamagnetic particles (SPP) complex
As one can see from the results, once the washing step was incorporated into the experiment design, no emitted light was observed from the sample, which allows us to conclude that no significant amount of chemicals were carried over
Summary
Deoxyribonucleic acid (DNA) molecules carry the majority of genetic information for all living species as well as viruses. A number of methods have been implemented in commercial DNA sequencers[2,7] and they are capable of rapidly sequencing the whole genome. These are routinely used in microbial,[8,9,10] DNA methylation,[11] and single nucleotide polymorphism (SNP) analyses.[12] these systems are complex, typically bulky, and require qualified personnel to operate them
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