2000 — 2003 |
Russo, James Ju, Jingyue Waarburton, Dorothy |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biophotonics: Combinatorial Fluorescent Energy Transfer Tags and Their Application For Multiplex Genetic Analyses
0086933 Ju The objective of the proposed research is to design and synthesize a large library of combinatorial fluorescent energy transfer (CFET) tags with unique fluorescence emission signatures that can be analyzed using a simple optical system. A fundamental limitation in fluorescent genetic analysis is the availability of fluorescent tags with distinctive fluorescence emissions. The PIs will develop a novel fluorescent-labeling scheme that uses a limited number of fluorescent molecules to create a large number of CFET tags with unique fluorescence signatures. The approach is based on the fluorescence energy transfer principle, the combinatorial concept, and that the energy transfer efficiency is dependent on the separation distance between the donor and acceptor. Synthetic procedures will be developed to construct a library of at least 20 of these CFET tags, and the spectroscopic properties of the CTET tags will be evaluated. The CFET tags will be generated using combinations of a number of different fluorescent molecules that will be linked through a rigid polymeric scaffold. The scaffold will be assembled by solid phase synthesis and solution coupling chemistry. The fluorescent signatures and chemical properties of the CFET tags will be carefully characterized to identify the most useful members of the collection. The tags will have particular utility in the massive high-throughput approaches of the genomics revolution, including DNA sequencing, identification of individual variations in the genome that are responsible for human disease, detection of microbial pathogens, and even drug screening, essentially any approach that can benefit by multiplexing operations. The utility of this library of CFET tags with unique fluorescence emission signatures will be validated by labeling a multiplex set of oligonucleotides for genetic mutation detection in a model system, as well as for genome-wide chromosome analyses.
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0.984 |
2001 — 2004 |
Turro, Nicholas (co-PI) [⬀] Ju, Jingyue |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Fluorescence Imaging Chip System For Massive Parallel Dna Sequencing
0097793 Ju The objective of this research is the development of a fluorescence imaging system for massive parallel DNA sequencing. This sequencing system includes the construction of a chip with immobilized single stranded DNA templates that can self prime for the generation of the complementary DNA strand in the polymerase reaction. The system also includes 4 unique fluorescently labeled nucleotide analogues with capped by a small chemical moiety to allow efficient incorporation into the growing strand of DNA as terminators in the polymerase reaction. A 4-color fluorescence imager is then used to identify the sequence of the incorporated nucleotide on each spot of the chip. After removal of the dye photochemically and the capping group, the polymerase reaction proceeds to incorporate the next nucleotide analogue and detect the next base. High density (>10,000 spots per chip) single stranded DNA will be immobilized on a 4 cm. x 1 cm. glass chip in order to identify more than 10,000 bases after each cycle. After 100 cycles a million base pairs will be generated on one sequencing chip.
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0.984 |
2003 — 2007 |
Ju, Jingyue |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Genomic Approaches to Neuronal Diversity and Plasticity @ Columbia University Health Sciences
DESCRIPTION: (provided by applicant): The objectives of the proposed research are the development of new genomic technologies for massively parallel DNA sequencing and large-scale gene expression analysis from single living nerve cells, and application of these technologies to study neural functions. The research teams from Columbia University and the University of Florida will closely interact to develop three innovative genomic technologies:(i) Massively Parallel DNA sequencing Chip System for sequencing SAGE library from neurons; (ii) Nanoscopic DNA Arrays for global gene expression profiling at the level of individual cells and subcellular compartments, and (iii) Real-time monitoring of multiple mRNA species in living neurons and defined cellular microdomains with high spatial resolution and fast temporal resolution. Each of these technologies will be rigorously tested and validated using a model memory-forming network of Aplysia. The technologies will then be implemented to explore three fundamental brain mechanisms: (1) the molecular basis of neuronal identity, (2) the molecular signals controlling the formation of the precise pattern of interconnections, which underlie behavior and, (3) the molecular basis of synapse-specific neuronal plasticity and neuronal growth. Using identified neurons in networks of Aplysia as experimental models we will study the role of asymmetric mRNA distribution in integrative functions and phenotypes of eukaryotic cells. We will use a hierarchical design to achieve structural resolution of single-cell profiling in a descending fashion, where a parallel genomic and functional analysis within the same memory-forming networks will be performed in the scheme: single neuron to single axon to single synapse. The gene expression profiling will be correlated with functional imaging at functionally characterized neurons and synaptic terminals in a simple network during the memory consolidation. The combined approach based on genomics, photochemistry, nanoscience and engineering, biochemistry, and neuroscience will be used to understand how neurons and synapses operate in the context of learning and memory. The technologies developed and the biological discoveries made in the project will have broad impact and applications to study how genes regulate cellular and organism behavior on the scale from simpler nervous systems in invertebrates to the human brain.
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0.984 |
2004 — 2009 |
Ju, Jingyue |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
An Integrated System For Dna Sequencing by Synthesis @ Columbia Univ New York Morningside
[unreadable] DESCRIPTION (provided by applicant): The objective of the proposed research is to develop an integrated system for DNA sequencing by synthesis (SBS) using photocleavable fluorescent nucleotides. The SBS system includes the construction of a chip with immobilized single stranded DNA templates by site-specific coupling chemistry. These templates contain a self priming moiety to generate the complementary DNA strand in polymerase reaction using 4 photocleavable fluorescent nucleotides whose 3-prime-OH group is modulated to allow their efficient incorporation into the growing strand of DNA as temporary terminators in the polymerase reaction. A 4-color fluorescence imager is then used to identify the sequence of the incorporated nucleotide on each spot of the chip. Upon removal of the fluorophore photochemically and reactivation of the 3-prime-OH group, the polymerase reaction will proceed to incorporate the next nucleotide analogue and detect the next base. It is estimated that 10,000 bases will be identified after each cycle on one sequencing chip that contains 10,000 different DNA templates. The engineering part of the proposal will pursue the development of two generations of SBS prototypes. The first generation system, Gen 1, will utilize conventional thermal cyclers, slides, spotting systems and confocal scanners to assemble reactions and automatically read out the fluorescent signals as bases are extended along the DNA templates. This configuration will allow the optimization of the chemistry, fluidics and photolysis processes to generate data using the SBS method relatively quickly. In parallel with the Gen 1 chip-based prototype development, we will develop several of the key instrumentation technologies for the second generation system, Gen 2, including an ultra-high throughput, low volume PCR/spotting system and a CCD camera fluorescence measurement system. A Gen 2 prototype system will be designed and constructed with the capability of producing sequence data from about 500,000 samples in parallel. The success of this approach will allow the development of a high-throughput DNA sequencing system for genome sequencing and resequencing. [unreadable] [unreadable]
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0.984 |
2005 — 2009 |
Ju, Jingyue |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gene Expression Analysis of Aplysia Neural Network @ Columbia Univ New York Morningside
DESCRIPTION (provided by applicant): Aplysia californica has been one of the dominant invertebrate model organisms utilized for research on the neurobiology of behavior, learning, and long-term memory. A key finding in recent studies is that both the functional and structural changes for memory storage are synapse-specific and require local translation. To understand the molecular mechanisms underlying long-term memory storage including its structural underpinning, it will be essential to determine how the population of mRNAs is destined for translation in synapses during memory formation. To examine this issue, we will extract mRNAs from kinesin transport complexes from ganglia and cultured individual sensory and motor neurons following treatment with the neurotransmitter serotonin (involved in learning-related long-term facilitation) and with FMRFamide (involved in long-term inhibition). At several time points after treatment, we will also characterize the mRNAs being actively translated on free and membrane-bound polysomes. The latter should include proteins targeted to the synaptic vesicles and membranes. Isolated mRNAs from all 3 populations (kinesin complexes, free polysomes, bound polysomes) will be hybridized to an Aplysia cDNA microarray containing features specific for the majority of central nervous system (CNS) genes. This microarray, an expansion of our current array, will be constructed from the unique clones in the EST libraries we have already generated from individual neurons, pedal-pleural ganglia, and the whole CNS of Aplysia. While we estimate these clones cover nearly 80% of all genes expressed in Aplysia neurons, only a fraction of the library has been annotated due to insufficient coding information. Nearly full annotation of the libraries will be achieved by sequencing full-length cDNAs from the same CNS sources. The full-length cDNA libraries and the Aplysia microarray will provide important resources for investigators to study learning and other behavior paradigms in this important model organism.
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0.984 |
2005 — 2007 |
Ju, Jingyue |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Modulating Nucleotide Size in Dna For Detection by Nanopore @ Columbia Univ New York Morningside
[unreadable] DESCRIPTION (provided by applicant): The goal of the proposal is to design and synthesize modified nucleotides to increase their size difference for single molecule DNA analysis by nanopores. We will pursue the following aims to study the feasibility of this approach: (1) Use solid phase synthesis to prepare single stranded DNA consisting of nucleotides carrying different sized modification groups and test these modified DNAs using nanopores to evaluate the parameters that are required to generate distinct blockade signals from each nucleotide in the DNA; (2) With knowledge gained in aim 1, design and synthesize modified nucleotides carrying different size groups for synthesis of modified DNAs in polymerase reaction. The single stranded DNA will then be detected using nanopores to search for condition to guide the design and modification of the nucleotides to achieve distinct blockade signals; (3) Design and synthesize nucleotides carrying small functional groups as hooks for DNA polymerase reaction to generate hook-labeled DNA products. Due to the small size of the hook, these nucleotides are expected to be good substrates for commonly used DNA polymerase to produce DNA products carrying the hook. The single stranded DNA products carrying the hook will then be isolated and selectively reacted with several different large functional groups to increase the size difference among the nucleotides in DNA. This DNA strand with the modified nucleotides will then be detected distinctly by nanopores to produce sequence data. The molecular tools developed here will facilitate achieving the long-term goal of single molecule sequencing by nanopores at single base resolution. [unreadable] [unreadable]
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0.984 |
2007 — 2008 |
Ju, Jingyue |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
3'-O-Modified Nucleotide Reversible Terminators For Pyrosequencing @ Columbia Univ New York Morningside
[unreadable] DESCRIPTION (provided by applicant): Pyrosequencing in a miniaturized device has been shown to have wide applications in genome sequencing. However, pyrosequencing with natural nucleotides has inherent difficulty in accurately deciphering the homopolymeric regions of DNA templates. The goal of this proposal is to design and synthesize a library of reversible nucleotide terminators to address this issue. The following three aims will be pursued: (1) Design and synthesis of four 3'-O-allyl-labeled nucleotides corresponding to A, C, G, T, as reversible terminators for pyrosequencing. We have produced small quantity of four 3'-O-allyl-labeled nucleotides in sufficient purity and have demonstrated that they can be used for pyrosequencing to produce accurate sequencing data in homopolymeric regions of DNA. We will further optimize the enzymatic conditions to increase the read length of pyrosequencing using the 3'-O-allyl-labeled nucleotides; (2) Design and synthesis of photocleavable 3'-O-modified nucleotide reversible terminators to compare with the 3'-O-allyl-labeled nucleotides to further optimize the pyrosequencing platform in terms of accuracy and readlength; (3) Evaluation of different micron-sized beads to immobilize the DNA template that are compatible to photocleavage or chemical cleavage conditions to perform pyrosequencing using the optimized set of reversible nucleotide terminators developed above. The molecular tools developed here will facilitate the optimization of pyrosequencing for de novo genome sequencing with unparalleled accuracy for biological and biomedical applications. [unreadable] [unreadable] [unreadable] [unreadable]
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0.984 |
2008 — 2009 |
Ju, Jingyue |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dna Sequencing With Reversible Dntp and Cleavable Fluorescent Ddntpterminators @ Columbia Univ New York Morningside
DESCRIPTION (provided by applicant): DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a new paradigm to decipher DNA sequences. In the grant application, we will pursue the development of a DNA sequencing system that is a hybrid between Sanger dideoxy chain terminating reaction and SBS using molecular engineering approaches. In this approach, four nucleotides, modified as reversible terminators (RTs, 3'-O-R1-dNTPs) by capping the 3'-OH with small reversible moiety (-R1) so that they are still recognized by DNA polymerase as substrates, are used in combination with a small amount of four cleavable fluorophore labeled dideoxynucleotide permanent terminators (PTs, ddNTPs-R2-fluorophore) to perform SBS on a DNA chip. DNA sequences will be determined by the unique fluorescence emission of each fluorophore on the ddNTPs. Upon removing the 3'-OH capping group on the RTs and the fluorophore from the PTs, the polymerase reaction will reinitiate and the DNA sequence can be continuously determined. We have recently demonstrated the feasibility of generating good quality sequencing data using this method. We will further develop this new method so that it can be readily used in the new generation of DNA sequencing by synthesis systems based on fluorescence detection. In addition, we will develop a walking strategy for SBS to use the immobilized DNA templates multiple times to increase the readlength of the SBS. We anticipate that up to 100 bp of continuous sequences will be produced by this approach, which can be used to pursue a variety of biomedical research projects.
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0.984 |
2008 — 2016 |
Yuste, Rafael (co-PI) [⬀] Heinz, Tony (co-PI) [⬀] Hielscher, Andreas (co-PI) [⬀] Ju, Jingyue Shepard, Kenneth [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Igert: Optical Techniques For Actuation, Sensing, and Imaging of Biological Systems
Progress in the biological sciences and medicine relies increasingly on methods, approaches, and strategies derived from synergistic interactions with the physical sciences and engineering. One notable example of this is the use of optical methods for biosensing and bioimaging. Furthermore, the tremendous nanoscale device fabrication capabilities built up in microelectronics and photonics furnish unparalleled opportunities for leveraging highly integrated platforms for on-chip biological sensor systems. By their nature, these applications cross through multiple disciplines and require a team with diverse expertise in the fundamental light/tissue interaction, complex optical instrumentation and imaging tools, and relevant biological systems. In this Integrative Graduate Education and Research Training (IGERT) program a new generation of scientists and engineers will be trained through a set of five research thrusts that cross three fundamental core competency areas: optics, photonics, and sensor electronics; biomolecular detection and cellular-level analysis; and applications to medicine and public health. Each IGERT trainee will be empowered to work at the boundaries between the disciplines and will be uniquely capable of contributing to advancements in this important emerging field. With 19 faculty members representing academic departments across Columbia University's School of Engineering and Applied Science, School of Arts and Sciences, Mailman School of Public Health, College of Physicians and Surgeons, and Teachers College, and incorporating strong interaction with City College, Queens College, and The Cooper Union in New York City, the IGERT trainees will experience a truly diverse community sharing in the integrated educational and research activities and will be exposed to a wide spectrum of cutting-edge applications. An external advisory board including industrial and government labs will provide additional connections between the IGERT and outside partners. Educationally, this IGERT program fulfills a compelling need to train a diverse workforce of U. S. scientists and engineers trained in an area of large and growing competitive importance to the United States. The proposed enrichment program provides IGERT fellows with enhanced training through experience in industry and government laboratories, seminars on professional development, career guidance, entrepreneurship, and discussion of ethical issues. Significant resources are committed to ensuring recruitment and retention of fellows from underrepresented groups. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.
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0.984 |
2008 — 2011 |
Ju, Jingyue |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Engineering Approach to Study Long Term Synaptic Plasticity @ Columbia Univ New York Morningside
DESCRIPTION (provided by investigator): The objectives of the proposed research are the development of new molecular engineering technologies for large-scale gene expression analysis from single neurons, and applications of these technologies to identify and characterize genes that are involved in long-term synaptic plasticity and growth. We will combine research expertise in Chemistry, Engineering and Biology to pursue the research and development of the following new molecular engineering approaches: (i) Massive Parallel DNA Sequencing Chip System for digital gene expression analysis from single cells and cell compartments;and (ii) Novel Molecular Probes for Real-time monitoring of multiple mRNA species in living neurons and defined cellular microdomains. Each of these technologies will be rigorously tested and validated using the simpler memory-forming network of Aplysia, a unique model organism for neurobiology. As a "proof-of concept", we will focus on using these approaches for the identification of gene-regulatory networks underlying the learning-induced synaptic growth. Specifically, we will characterize a molecular cascade of events induced by serotonin, leading to the formation of new synapses and a long-term enhancement of synaptic strength also known as cellular manifestations of learning and memory mechanisms. The long-term goal of this project is to implement these new technologies to explore two fundamental brain mechanisms: (1) the molecular basis of neuronal growth;(2) the molecular signals controlling synapse-specific neuronal plasticity. Using the sensory neurons of the neuronal networks in Aplysia as an experimental model, we will study the role of asymmetric mRNA distribution in integrative functions and phenotypes of eukaryotic cells. We will use a hierarchical design to achieve structural resolution of single-cell profiling in a descending fashion, where a parallel genomic and functional analysis will be performed according to the following scheme: single neuron->single axon->single synapse. The gene expression profiling will be validated using a set of complementary approaches, correlated with functional imaging of selected mRNAs at functionally characterized neurons and synaptic terminals during various stages of 5-HT induced synaptic growth. The combined approach based on Chemistry, Engineering, and Neuroscience will be used to understand how neurons and synapses operate in the context of learning and memory. The technologies developed and the biological discoveries made in the project will have a broad impact in deciphering the molecular mechanisms of neurological disorders.
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0.984 |
2009 — 2011 |
Ju, Jingyue |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single Molecule Dna Sequencing by Fluorescent Nucleotide Reversible Terminators @ Columbia Univ New York Morningside
DESCRIPTION (provided by applicant): The ability to sequence a human genome with high accuracy and speed, and at low cost, is critical to the emerging field of personalized medicine. In response to this demand, our research team developed the novel method of DNA sequencing-by-synthesis (SBS) on a solid surface, which has been recognized as a successful new paradigm for deciphering DNA sequences. In this grant application, we will use molecular engineering approaches to take our successful SBS strategy to the next level by adapting it for single molecule sequencing using fluorescent reversible terminators. Template DNA molecules will be attached to a glass surface modified by covalent attachment of PEG-primers under conditions where as many as 1 billion clearly separated single molecules are attached to the slide and their location registered by the presence of a cleavable fluorescent moiety. SBS will then be conducted using reversible blocked nucleotides with an appropriate set of cleavable fluorophores. We have also developed a walking strategy that permits re-use of the template multiple times to increase SBS readlength. We will modify a TIRF microscope to create a device with an enhanced microfluidic flow cell platform to permit large-scale detection of single molecules during each cycle of SBS. Finally, we have designed a number of DNA library construction methods that avoid amplification and a paired-end sequencing strategies compatible with the single molecule SBS approach. This will permit us to test the system with real genomic DNA, cDNA and other templates from ongoing biomedical research collaborations. With a billion DNA templates immobilized on a chip at single molecule resolution, even 30 to 50 base reads will cover the entire human genome at good coverage on a single chip. Public Health Relevance: The realization of the need for personalized medicine has encouraged the development of technologies able to sequence the human genome with high accuracy and speed at low cost. To approach this goal, we have combined the concepts of our successful sequencing by synthesis and sequence walking method with the ability to utilize single molecules. The latter avoids the necessity of cloning or otherwise amplifying DNA before sequencing, which is in fact one of the most expensive and time consuming parts of the process, and can lead to undesirable biases in the DNA sequences. With a billion DNA molecules immobilized on a chip at single molecule resolution, even read lengths of 30 or 50 bases will provide the ability to sequence the entire human genome at high accuracy on a single sequencing chip.
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0.984 |
2012 — 2016 |
Ju, Jingyue Kandel, Eric R (co-PI) [⬀] Moroz, Leonid L (co-PI) [⬀] Sander, Chris (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genomic Approaches to Deciphering Memory Circuits @ Columbia Univ New York Morningside
DESCRIPTION (provided by applicant): The objective of the proposed research is to conduct a thorough single-cell and cell-compartment gene expression study through the application of high throughput genomic technologies to identify the genomic bases of neuronal identity, polarity and plasticity. Utilizing the well-studied gill withdrawal reflex memory circuit from the model organism Aplysia californica, our goal is to define systematically the molecular repertoire (genomic blueprint) of the neurites and individual synapses of the key neurons that make up this cellular ensemble. We will define the compartmental transcriptomes (the sets of mRNAs, miRNAs and other ncRNAs) within the components of the functional circuit (cells and synapses), which are reconstituted in vitro by co- culture of 2-4 of its best characterized cells (L7 motor neuron, sensory neuron, stimulatory and inhibitory interneurons). This fully operational neural circuit reconstructed in cell culture bears many important properties of the intact circuit, and has been used with great success to ascertain the molecular underpinnings of memory formation in Aplysia, numerous aspects of which are conserved within the animal kingdom, including in the human brain. The systems biology approach will be applied to reveal gene regulatory networks and their potential role in the establishment and maintenance of long-term memory using learned fear as an experimental paradigm, focusing on synaptic mechanisms of long-term facilitation (LTF) and depression (LTD). We will use this genomic and systems biology approach to explore the following three fundamental brain mechanisms: (1) the molecular basis of neuronal identity, by revealing those transcripts that are unique to or shared among these neurons or specialized synapses; (2) the molecular signals controlling cellular polarity and the formation of the precise pattern of interconnections which underlie behavior, in part directed by the distribution of mRNAs in the central and peripheral compartments of these cells; and (3) the molecular basis of synapse-specific neuronal plasticity and neuronal growth, with special attention paid to the mRNA repertoire within the individual synapses at the junctions between pairs of pre- and post-synaptic neurons. The combined approach will take advantage of an already established team of experts in genomics, bioengineering, neuroscience, and bioinformatics. Though these paradigms will be established in the large well-characterized neurons of Aplysia, the mechanisms revealed and the technologies developed will have a broad impact in the biology of any polarized cell type with asymmetric distribution of RNAs and proteins.
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0.984 |
2013 — 2015 |
Church, George M Ju, Jingyue Russo, James John |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
An Integrated System For Single Molecule Electronic Sequencing by Synthesis @ Columbia Univ New York Morningside
There is a great need to reduce the cost of DNA sequencing to achieve the goal of the $1000 genome. We recently developed a new nanopore-based sequencing by synthesis (Nano-SBS) approach. In this project, we will pursue the development of the Nano-SBS approach into a high throughput real-time single-molecule sequencing platform. In the Nano-SBS method, a polymer tag of distinct size and charge is attached to the terminal phosphate of each of the four nucleotides. When the complementary nucleotide analog enters a template-primer-polymerase complex that is attached to the nanopore during the polymerase reaction, the tag specific for that nucleotide is captured in the voltage gradient within the nanopore and results in a current blockade unique to each tag for sequence determination. The polymerase is covalently attached to the nanopore by a short linker so the polymeric tag will have sufficient time to enter the vestibule and constriction of the nanopore prior to its release ensuring that its current blockade signal is recorded by the nanopore. The extended DNA strand bears only natural nucleotides, enabling long reads. We have carried out the key proof-of-principle experiments to demonstrate the feasibility of this approach. Here our strong team of nucleic acid chemists, genomic scientists, electrical engineers, and nanofabrication experts will further develop the Nano-SBS as a high throughput genomic sequencing system. We will develop robust methodology to attach polymerase to the .-hemolysin (AHL) nanopore and synthesize nano-tags with unique chemical properties resulting in AHL current blockades distinct from each other and nucleotide precursors. We will test these elements in single pores as well as in new nanopore array chips with separate sensors and circuits for each pore. We will produce mutant AHL and polymerase constructs and link them to each other, selecting for the combination that assures accurate DNA extension reactions, and rapid capture and detection of tags in nanopores. The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques. We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system. These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine. 1
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0.984 |
2020 |
Church, George M Ju, Jingyue Kalachikov, Sergey |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Novel Tagged Nucleotides and Nanopore Constructs @ Columbia Univ New York Morningside
Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Tagged Nucleotides and Nanopore Constructs With past NIH funding, we developed a single molecule nanopore-based sequencing by synthesis (SBS) strategy (Nanopore SBS) that accurately distinguishes the four DNA bases by electronically detecting 4 different polymer tags attached to the 5?-phosphate-modified nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. 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. 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. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primed template and polymerase, the polymer 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 distinguishable 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. While the Nanopore-SBS approach already produces good quality sequences, further optimization and development are needed to increase sequencing accuracy, while maintaining the ability of our nanopore-based single-molecule electronic system to produce long reads in real time. In this proposal, we will design and synthesize novel tagged nucleotides and construct nanopore-polymerase conjugates to control the sequencing reaction speed and increase single-molecule sequencing accuracy substantially, achieving desired polymerase catalytic rates and more efficient and consistent tag capture by the pores. We will use high ratios of unincorporable-to-incorporable tagged nucleotides to perform Nanopore-SBS. This will provide ample time to register currents due to the 4 unique tags on the unincorporable A, C, G and T nucleotides which display template-dependent binding to the polymerase ternary complex but are not incorporated into the growing DNA strand, followed by a new current level due to a 5th tag on the incorporable nucleotide which marks the transition to the extension step. This effectively eliminates insertion and deletion sequence artifacts, increases accuracy, and will be especially advantageous in DNA homopolymer repeat regions. This approach allows detection of a single nucleotide binding event multiple times (stutters) before the actual incorporation event, overcoming the inherent limitation of single molecule detection methods that only allow one chance for measurement. After optimizing the system with synthetic DNA templates, circular DNA libraries will be generated from viral and bacterial genomes to test this sequencing approach. With the improved tagged nucleotides, better regulated reaction kinetics, and newly designed polymerase-pore complexes, we will test the accuracy of our system on the nanopore arrays by sequencing these libraries at high coverage and comparing the results with other sequencing systems.
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0.984 |