Northeastern University Background
DNA sequencing technologies have undergone tremendous improvements in recent years in throughput and quality. One advantage of single-molecule DNA sequencing technologies is that long DNA molecules can be read in a single run, which greatly facilitates genome assembly and aids in the construction of reference-quality genomes. In 2003, Pacific Biosciences introduced a version of single-molecule sequencing called single molecule, real-time (SMRT) sequencing, in which DNA replication by a polymerase is monitored in real-time by tracking the incorporation of fluorescently labeled nucleotides of different colors. This method offers long reads (>15,000 bp) and the ability to detect epigenetic modifications by tracking delays in the polymerase kinetics of incorporation of different nucleotides.
However, although the throughput and accuracy of SMRT-sequencing have vastly improved in recent years, loading efficiency and length bias are both still problematic. This translates to high DNA input requirements (>100 ng) and length-purified libraries in order to alleviate bias towards reading short DNA lengths.
In the proposed invention, Northeastern University researchers introduce an alternative way to capture DNA molecules into ZMWs that does not require the use of a freestanding membrane. This device is called an electrochemical lasso. The working principle of this design relies on embedding metallic electrodes under the waveguides to create an electric field. The application of a voltage to the electrode layer with the use of proper electrolyte allows efficient electrophoretic DNA capture at picogram levels. To do so, a thin metallic film (MLasso) is deposited on a substrate (S) which serves as an electrode. This electrode is insulated from a metallic ZMW layer (MZMW) using a dielectric spacer. This device eliminates the need for free-standing membranes, and therefore is mechanically stable, allows scaled-up fabrication, reduces the background optical noise, and improves the DNA loading efficiency by several orders of magnitude.
High capture efficiency
Reduced fabrication costs
Accurate radial positioning of the enzyme probed in the single-molecule assay