In the last two decades, there has been great interest in taking advantage of nanopores, naturally occurring or solid-state ion channels, for polymer characterization and distinguishing the bases of DNA in a low-cost, rapid, single-molecule manner ( 6– 9). Although optical methods for single-molecule sequencing have been achieved and commercialized, the most successful, Pacific Biosciences’ single molecule real-time (SMRT) sequencing by synthesis (SBS) approach, requires expensive instrumentation and the use of fluorescently tagged nucleotides ( 4, 5). Single-molecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias ( 3, 4). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete ( 1, 2). Information on individuals’ genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner ( 1). It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. The importance of DNA sequencing has increased dramatically from its inception four decades ago. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences.
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