Pyrosequencing on a glass surface

Pyrosequencing on a glass surface

WoS Article

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.

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.

DNA sequence determination; pyrosequencing; droplets; magnetic particles; open-surface microfluidics; virtual reaction chambers

DNA sequence determination; pyrosequencing; droplets; magnetic particles; open-surface microfluidics; virtual reaction chambers

ALMEIDA, A.; MANZ, A.; NEUŽIL, P.

2017

16.02.2016

Royal Society of Chemistry

Londyn

1473-0197

Lab on a chip

16

6

United Kingdom of Great Britain and Northern Ireland

1063

1071

9

https://pubs.rsc.org/en/content/articlelanding/2016/LC/C6LC00114A

http://hdl.handle.net/11012/203127

C6LC00114A_pyrosequencing
c6lc00114a