Single-molecule nanopore DNA sequencing by synthesis (SBS) involves the identification of distinctly tagged DNA nucleotides as the tags pass through the pore of an ion channel during enzyme-catalyzed template-directed DNA synthesis.
To hone the nanopore SBS technique, researchers fashioned a nanopore array composed of α-hemolysin channels that were coupled to a DNA-synthesizing enzyme with high staying power, bound to a primed DNA template, and embedded in lipid bilayers on an electronic chip bearing multiple electrodes. The addition of distinct custom-designed polymer-tagged nucleotides to the nanopore array triggers DNA synthesis.
By blocking the channel's ionic current to different extents, the tags provide a readout of the template sequence in real time with single-base resolution; after each catalytic step, the tags exit the pore and end the current blockade, allowing continued DNA synthesis that enables sequence determination. Tethering the sequencing enzyme to nanopores and arraying nanopore sensors on the chip together enabled simultaneous, rapid, and high-resolution sequencing of long stretches of multiple DNA strands.
Though the precise sequencing rate and accuracy of the improved technique remain to be established, the authors suggest that the method might represent a prototype high-throughput sequencing approach with applications in precision medicine, pending further improvements. Such advances may include improved DNA-synthesizing enzymes and integrated circuits with increased number of electrodes, according to the authors.
http://www.pnas.org/content/early/2016/04/14/1601782113
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