Researchers from Columbia University,
with colleagues at Genia Technologies (Roche), Harvard University and
the National Institute of Standards and Technology (NIST) report
achieving real-time single molecule electronic DNA sequencing at
single-base resolution using a protein nanopore array.
DNA sequencing is the key technology for personalized and precision medicine
initiatives, enabling rapid discoveries in biomedical science. An
individual's complete genome sequence provides important markers and
guidelines for medical diagnostics, healthcare, and maintaining a
healthy life. To date, the cost and speed involved in obtaining highly
accurate DNA sequences has been a major challenge. While various
advancements have been made over the past decade, the high-throughput
sequencing instruments widely used today depend on optics for the
detection of four DNA building blocks: A, C, G and T. To explore
alternative measurement capabilities, electronic sequencing of an
ensemble of DNA templates has been developed for genetic analysis.
Nanopore strand sequencing, wherein a single strand DNA is threaded
through the nanoscale pores under an applied electrical voltage to
produce electronic signals for sequence determination at single molecule
level, has recently been developed; however, because the four
nucleotides are very similar in their chemical structures, they cannot
easily be distinguished using this method. Researchers are therefore
actively pursing the research and development of an accurate
single-molecule electronic DNA sequencing platform as it has the
potential to produce a miniaturized DNA sequencer capable of deciphering
the genome to facilitate personalized precision medicine.
A team of researchers at Columbia Engineering, headed by
Jingyue Ju (Samuel Ruben-Peter G. Viele Professor of Engineering,
Professor of Chemical Engineering and Pharmacology, Director of the
Center for Genome Technology & Biomolecular Engineering), with
colleagues at Harvard Medical School, led by George Church (Professor of
Genetics); Genia Technologies, led by Stefan Roever (CEO of Genia); and
John Kasianowicz, the Principal Investigator at NIST, have developed a
complete system to sequence DNA in nanopores electronically at single
molecule level with single-base resolution. This work, entitled,
"Real-Time Single Molecule Electronic DNA Sequencing by Synthesis Using
Polymer Tagged Nucleotides on a Nanopore Array," is published in the
journal, Proceedings of the National Academy of Sciences (PNAS) Early Edition.
Previously, researchers from the laboratories of Ju at
Columbia and Kasianowicz at NIST reported the general principle of
nanopore sequencing by synthesis (SBS), the feasibility of design and
synthesis of polymer-tagged nucleotides as substrates for DNA
polymerase, the detection and the differentiation of the polymer tags by
nanopore at the single molecule level [Scientific Reports 2, 684 (2012) DOI: 10.1038/srep00684]. The current PNAS
paper describes the construction of the complete nanopore SBS system to
produce single molecule electronic sequencing data with single-base
resolution. This SBS strategy accurately distinguishes four DNA 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 polymerase, a
DNA-synthesizing enzyme. The researchers designed and synthesized novel
nucleotides tagged at the terminal phosphate with oligonucleotide-based
polymers to perform nanopore SBS on an α-hemolysin protein nanopore
array platform. The tags on the polymer-labeled nucleotides, which were
verified to be active substrates for DNA polymerase, produce different
electrical current blockade levels. They constructed a nanopore array on
an electronic chip bearing multiple electrodes; the array is composed
of protein channels that were coupled to a DNA polymerase that was bound
to a primed DNA template. Addition of distinct custom-designed polymer
tagged nucleotides to the nanopore array triggers DNA synthesis. By
blocking the channel's ionic current to different levels, the distinct
tags provide a readout of the template sequence in real time with
single-base resolution.
As Carl Fuller, lead author, Adjunct Senior Research Scientist in the
Ju Laboratory of the Chemical Engineering Department at Columbia and
Director of Chemistry at Genia, points out, "The novelty of our nanopore
SBS approach begins with the design, synthesis, and selection of four
different polymer-tagged nucleotides. We use a DNA polymerase covalently
attached to the nanopore and the tagged nucleotides to perform SBS.
During replication of the DNA bound to the polymerase, the tag of each
complementary nucleotide is captured in the pore to produce a unique
electrical signal. Four distinct polymer tags yielding distinct
signatures that are recognized by the electronic detector in the
nanopore array chip are used for sequence determination. Thus, DNA
sequences are obtained for many single molecules in parallel and in real
time. The four polymer tags are designed to offer much better
distinctions among themselves, in contrast to the small differences
among the four native DNA nucleotides, thereby overcoming the major
challenge faced by other direct nanopore sequencing methods." Moreover,
the tags can be further optimized with respect to size, charge, and
structure to provide optimal resolution in the nanopore SBS system."This exciting project brings together scientists and engineers from both academia and industry with combined expertise in molecular engineering, nanotechnology, genomics, electronics and data science to produce revolutionary, cost-effective genetic diagnostic platforms with unprecedented potential for precision medicine," says Ju. "We are extremely grateful for the generous support from the NIH that enabled us to make rapid progress in the research and development of the nanopore SBS technology, and the outstanding contributions from all the members of our research consortium."
According to Ju, the researchers have already pushed beyond what was demonstrated in the PNAS
study where the sequencing data was obtained on an early prototype
sequencer based on nanopore SBS. The throughput and performance of the
current sequencer has progressed beyond what was reported in the PNAS
paper. The feasibility of reaching read-lengths of over 1000 bases of
DNA has recently been achieved. Going forward, the collaborative
research team will continue to optimize the tags by tweaking the
linkers, structure, and charge at the molecular level, and fine tuning
the polymerase and the electronics for the nanopore SBS system with an
aim to accurately sequence an entire human genome rapidly and at low
cost, thereby enabling it to be used in routine medical diagnoses.
More information:
Carl W. Fuller et al, Real-time single-molecule electronic DNA
sequencing by synthesis using polymer-tagged nucleotides on a nanopore
array, Proceedings of the National Academy of Sciences (2016). DOI: 10.1073/pnas.1601782113
Post a Comment