UC San Diego

The goal of this work is to develop an experimental platform to carry out controlled DNA denaturation reactions while monitoring the fractional hybridization using fluorescently labeled oligonucleotides. Because shorter oligonucleotides denature at a lower temperature, it is possible to decode the sequence based on their melting temperatures. This technique, known as Sequencing by Denaturation, is based on the sequential denaturation of DNA fragments generated by a Sanger dideoxy sequencing reaction on surface-bound DNA templates. To perform these experiments, a device was constructed consisting of nine microfluidic channels formed by an adhesive silicone gasket sandwiched between a coverslip and a stainless steel plate that is encased in an aluminum block. Cooling and heating capabilities are provided by thermoelectric modules, which are in contact with another aluminum block that serves as the heatsink and microscope stage insert. The device satisfies the major criteria for performing this sequencing method. The microfluidic channels are uniformly heated to ensure that all the molecules experience the same temperature. Additionally, the device undergoes a consistent thermal expansion throughout the temperature ramp, allowing the imaging plane to be tracked by the microscope. We demonstrated the ability to determine the components of a denaturation profile created by the combination of four different oligonucleotide lengths. Based on simulation data, we also determined that it might be possible to resolve a denaturation curve using as few as fifty probes. Due to the speed, simplicity, and low cost of sequencing with SBD, it has potential to enable large-scale genome re-sequencing and genotyping