A new technology for the synthesis of long DNA strings (original) (raw)
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Recent Patents of Nanopore DNA Sequencing Technology: Progress and Challenges
Recent Patents on DNA & Gene Sequences, 2010
DNA sequencing techniques witnessed fast development in the last decades, primarily driven by the Human Genome Project. Among the proposed new techniques, Nanopore was considered as a suitable candidate for the single DNA sequencing with ultrahigh speed and very low cost. Several fabrication and modification techniques have been developed to produce robust and well-defined nanopore devices. Many efforts have also been done to apply nanopore to analyze the properties of DNA molecules. By comparing with traditional sequencing techniques, nanopore has demonstrated its distinctive superiorities in main practical issues, such as sample preparation, sequencing speed, cost-effective and read-length. Although challenges still remain, recent researches in improving the capabilities of nanopore have shed a light to achieve its ultimate goal: Sequence individual DNA strand at single nucleotide level. This patent review briefly highlights recent developments and technological achievements for DNA analysis and sequencing at single molecule level, focusing on nanopore based methods.
DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. 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 po-lymerase. 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. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active sub-strates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were com-plexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. 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. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly dis-tinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. 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. single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. 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. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome se-quencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). 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). 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. Single-molecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). 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). 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). Three nanopore sequencing approaches have been pursued: strand se-quencing in which the bases of DNA are identified as they pass sequentially through a nanopore (6, 7), exonuclease-based nanopore Significance Efficient cost-effective single-molecule sequencing platforms will facilitate deciphering complete genome sequences, determining haplotypes, and identifying alternatively spliced mRNAs. We demonstrate a single-molecule nanopore-based sequencing by synthesis approach that accurately distinguishes four DNA bases by electronically detecting and differentiating four different polymer tags attached to the terminal phosphate of the nucleotides during their incorporation into a growing DNA strand in the polymerase reaction. With nanopore detection, the distinct polymer tags are much easier to differentiate than natural nucleotides. After tag release, growing DNA chains consist of natural nucleotides allowing long reads. Sequencing is realized on an electronic chip containing an array of independently addressable electrodes, each with a single polymerase–nanopore complex, potentially offering the high throughput required for precision medicine.
Sequencing ultra-long DNA molecules with the Oxford Nanopore MinION
2015
Oxford Nanopore Technologies' nanopore sequencing device, the MinION, holds the promise of sequencing ultra-long DNA fragments >100kb. An obstacle to realizing this promise is delivering ultra-long DNA molecules to the nanopores. We present our progress in developing cost-effective ways to overcome this obstacle and our resulting MinION data, including multiple reads >100kb.
Design and Characterization of Programmable DNA Nanotubes
Journal of The American Chemical Society, 2004
DNA self-assembly provides a programmable bottom-up approach for the synthesis of complex structures from nanoscale components. Although nanotubes are a fundamental form encountered in tilebased DNA self-assembly, the factors governing tube structure remain poorly understood. Here we report and characterize a new type of nanotube made from DNA double-crossover molecules (DAE-E tiles). Unmodified tubes range from 7 to 20 nm in diameter (4 to 10 tiles in circumference), grow as long as 50 µm with a persistence length of ∼4 µm, and can be programmed to display a variety of patterns. A survey of modifications (1) confirms the importance of sticky-end stacking, (2) confirms the identity of the inside and outside faces of the tubes, and (3) identifies features of the tiles that profoundly affect the size and morphology of the tubes. Supported by these results, nanotube structure is explained by a simple model based on the geometry and energetics of B-form DNA.
The potential and challenges of nanopore sequencing
Nature Biotechnology, 2008
A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of `third generation' instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.
Replication of individual DNA molecules under electronic control using a protein nanopore
Nature Nanotechnology, 2010
Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file order by an applied voltage. Here we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-hemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalyzed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80 mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond time scale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held atop the nanopore orifice at 80 mV applied potential. Sequential nucleotide additions by KF were observed upon controlled voltage reversals. * Correspondence and requests for materials should be addressed to [MA]. makeson@soe.ucsc.edu. Author Contributions. F.O. designed experiments and performed data analysis; K.R.L. co-authored the manuscript, designed and conducted experiments; S.B., G.M.C., and J.M.D. conducted nanopore experiments including FSM implementation; D.W.D. conceived the idea of coupling polymerases to nanopores and helped design experiments; M.A. co-authored the manuscript, conceived the blocking oligomer strategy, designed experiments, and is responsible for the overall quality of the work. Competing Financial Interests. M. Akeson and D. Deamer are consultants to Oxford Nanopore Technologies (Oxford, England).
Proceedings of the National Academy of Sciences, 2016
Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability o...
Synthetic Biology
Synthetic biology utilises the Design-Build-Test-Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilise this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using PCR is introduced and sequencing data is analysed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents iden...
A review on nanopore sequencing technology, its applications and challenges Mirza Jawad Ul Hasnain
Pure and Applied Biology
In this review, we describe another age of sequencing 'Nanopore Sequencing technology" which is rapidly growing to fulfill the gap in advancements, which hold the capacity for significantly larger perused read lengths, less time consuming and lower by expense. Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than 1000,andpossiblyforevenlessthan1000, and possibly for even less than 1000,andpossiblyforevenlessthan100.The need has never been more prominent for progressive advances that convey time efficient, reasonable and exact genome data. This quest has led to the advancements in the field of Next Generation Sequencing (NGS). The economical production of huge volumes of sequencing information is the essential advantage over traditional techniques. The preliminary methods of sequencing namely Sanger sequencing and Second Generation Sequencing provide the basis to the propelling and surprising number of logical advances. However, the evolving genomics and specifically the sequences sciences provide prolific basis for extra development in this space of research. We hereby ponder upon the broad range of Nano-pore sequencing techniques along with their applications in the emerging era of genomics and its advancement, also the word goes towards the importance of Nano-pore sequencing advancements in the emerging markets of sequencing like that of Pakistan.