DNA conjugation on functionalized plastic surfaces for sequential, iterative single molecule sequencing

We developed a method for repeated and sequential retrieval of arbitrary DNA elements. A click chemistry process was used to conjugate the DNA molecules onto a plastic surface within the interior of microcentrifuge tubes. For this study, we utilized synthetic DNA sequences that encode arbitrary data and designed PCR primers for amplification. Specifically, the DNA was tailed with trans-cyclooctene (TCO) and was then conjugated to plastic surfaces functionalized with methyltetrazine (MTz). The covalent DNA attachment to the plastic surface enables repeated and non-destructive polymerase-based copying and amplification of the original source molecules. In summary, we demonstrate a new type of DNA storage media with the property of long-term stability and ability to read different groups or files of DNA data. For this proof-of-concept study, we demonstrate the key features of this technology including: (1) characterization of the DNA conjugation process using control strands, (2) conjugation kinetics, and (3) amplification and sequencing of specific DNA data elements over multiple retrieval operations from the same plastic surface. Specifically, we showed that different DNA "file groups" with information could be accessed multiple times with single molecule sequencing. We also compared the performance metrics of PCR versus recombinase polymerase amplification (RPA). Our results indicate that RPA has better performance metrics in terms of reducing contamination and improved yield of retrieved data from across sequential experiments. Overall, we demonstrated a plastic-based DNA storage system for robust and reliable iterative, targeted DNA retrieval, and sequencing.