(337e) Microfabricated Microemulsion Droplet Generator Enables The Pcr Colony Front End Of The Microbead Integrated DNA Sequencer

The Microbead INtegrated DNA Sequencer (MINDS) project is advancing Sanger sequencing technology to its ultimate limit: the production of complete sequencing separations from a single template PCR colony by coupling bead-based PCR template amplification with integrated microfluidic extension and separation. As a part of this process we have recently shown that Sanger extension and sequencing separation can be effectively integrated by the use of an oligonucleotide affinity gel matrix for extension sample purification and concentration [1]. We have further demonstrated high quality sequencing reads from only 100 attomoles of PCR amplicon in a sample purification-CE microdevice that incorporates a more efficient inline injection system [2]. These advances now make it feasible to sequence directly from the PCR products generated by a single molecule in controlled size emulsions. To enable a high-throughput front end for attomole-scale Sanger DNA sequencing and other genetic analysis, we have now developed a microfabricated droplet generator [3] to rapidly form highly monodisperse, nanoliter volume droplets of PCR mix in emulsion oil. Around 2000, 5 nL droplets, formed this way, are collected in individual tubes and simultaneously temperature cycled using a block thermal cycler. The generator also provides a reliable way to introduce primer functionalized microbeads in the droplets, which act as substrates for solid phase PCR and facilitate extraction of amplified products from individual droplets. To evaluate this system, a 545 bp region of the pUC18 genome was successfully amplified on ~34 μm diameter beads contained in ~5 nL volume droplets. Sufficient sequencing template can be amplified from single DNA molecules on individual beads to enable Sanger extension and sequencing in the ultra-efficient integrated sequencing processors [1, 2]. The work presented here establishes the feasibility of the MINDS process that will take Sanger sequencing, which has the unique ability to produce long and accurate sequence reads, to its fundamental molecular limit.

REFERENCES

[1] ?Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing?, R.G. Blazej, P. Kumaresan, R.A. Mathies, Proc. Natl. Acad. Sci. USA, 103, 7240 (2006).

[2] ?Inline-injection microdevice for attomole-scale Sanger DNA sequencing?, R.G. Blazej, P. Kumaresan, S.A. Cronier, R.A. Mathies, Anal. Chem., In press.

[3] ?High-temperature microfluidic synthesis of CdSe nanocrystals in nanoliter droplets?, E.M. Chan, A.P. Alivosatos, R.A. Mathies, J. Am. Chem. Soc., 127, 13854 (2005).