Nanobiotechnology Projects
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Other Research Projects
PI: Prof. Ronald W. Davis Collaborators/Advisors Prof. Fabian Pease
Melike Abacioglu |
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Thermosequencing: A Novel Method of DNA sequencing
“Never has the state of DNA sequencing technology been in greater flux than today.”
Michael L. Metzker, Nature Reviews, Genetics 2008.
For the past three decades, Sanger’s method has been the primary DNA sequencing technology; however, inherent limitations in cost and complexity have limited its usage in personalized medicine and ecological studies. Some other methods such as Pyrosequencing or Solexa technology continue to reduce costs and increase throughput; however still the current technologies are not match with the need of individual genome sequencing enabling personalized medicine.
Our Thermosequencing is a novel sequencing technique with the potential to yield significant improvements in cost and read length; thermosequencing also has several distinct advantages over existing synthesis techniques: neither expensive fluorescent labels, reporting enzymes, nor optical detectors are required and native DNA polymerase may be used. Thus reagent and instrument costs are greatly reduced, while the long read-length capability of native DNA polymerase may be exploited. We have demonstrated proof-of-concept by detecting the heat of the nucleotide incorporation reaction using infrared microscopy and micro-calorimetry experiments. We have also validated the use of a microfluidic format through numerical modeling of the reaction and heat transfer.
Our aim in this project is to demonstrate thermosequencing of a 500 base pair template using a microfabricated temperature sensor, develop a microfluidic fluid control system, and fabricate an integrated thermosequencer prototype.
Fig.1: Schematic cartoon of a thermosequencer well with DNA-coated bead
Fig. 2: Enzymatic Models of Pyrosequencing vs. Thermosequencing [Esfandyarpour, H., et al. JVST. B: Microelectronics and Nanometer Structures, 26:2, pp 661-665].
Fig. 3: Simulation results of heat profile during the DNA incorporation.
Fig. 4: The time-dependent concentrations of dNTP and PPi at the well bottom are shown for the 2D simulation. The dNTP concentration asymptotically increases to equilibrate with the 3 mM dNTP concentration in the channel. The maximum PPi concentration at the well bottom is 0.1407 mM (time scale is in seconds) [Esfandyarpour, H., et al., J Biomicrofluidics, 2 (1)].
Fig. 5: Control channel schematics for the double _top left_ and single _bottom left_ control line systems. In both cases, pressurization of the control channels induces expansion for mass and thermal insulation of the well. The double-control line system for a 100-_m-wide channel is shown at right [Esfandyarpour, H., et al., J Biomicrofluidics, 2 (1)].
Fig. 6: The resulting concentrations of reaction products were used to calibrate the reaction speed and to design the heat sensors in the Thermosequencing technology [Esfandyarpour, H., et al., J Biomicrofluidics, 2 (1)].
