Synthetic DNA

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Andrew D Ellington - One of the best experts on this subject based on the ideXlab platform.

  • Synthetic DNA synthesis and assembly putting the Synthetic in Synthetic biology
    Cold Spring Harbor Perspectives in Biology, 2017
    Co-Authors: Randall A Hughes, Andrew D Ellington
    Abstract:

    : The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in Synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality Synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read-write cost gap.

  • Current Protocols in Molecular Biology - Enrichment of Error‐Free Synthetic DNA Sequences by CEL I Nuclease
    Current Protocols in Molecular Biology, 2012
    Co-Authors: Randall A Hughes, Aleksandr E. Miklos, Andrew D Ellington
    Abstract:

    As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences Synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made Synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of Synthetic biology. However, Synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from Synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of Synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment. Curr. Protoc. Mol. Biol. 99:3.24.1-3.24.10. © 2012 by John Wiley & Sons, Inc. Keywords: CEL I; error correction; Synthetic DNA; Surveyor nuclease; Synthetic biology; oligonucleotide; oligonucleotide synthesis

  • Enrichment of error-free Synthetic DNA sequences by CEL I nuclease.
    Current protocols in molecular biology, 2012
    Co-Authors: Randall A Hughes, Aleksandr E. Miklos, Andrew D Ellington
    Abstract:

    As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences Synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made Synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of Synthetic biology. However, Synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from Synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of Synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment.

  • Current Protocols in Molecular Biology - Design and assembly of large Synthetic DNA constructs.
    Current Protocols in Molecular Biology, 2012
    Co-Authors: Aleksandr E. Miklos, Randall A Hughes, Andrew D Ellington
    Abstract:

    The availability of custom Synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of Synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing “in-house” gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides. Curr. Protoc. Mol. Biol. 99:3.23.1-3.23.18. © 2012 by John Wiley & Sons, Inc. Keywords: gene synthesis; Synthetic genes; Synthetic DNA; Synthetic biology; protein expression; gene assembly; oligonucleotides; oligonucleotide assembly; oligonucleotide synthesis; inside-out nucleation; PCR

Michael R Wasielewski - One of the best experts on this subject based on the ideXlab platform.

  • dynamics of photoinduced charge transfer and hole transport in Synthetic DNA hairpins
    Accounts of Chemical Research, 2001
    Co-Authors: Frederick D Lewis, Robert L Letsinger, Michael R Wasielewski
    Abstract:

    The dynamics of photoinduced charge separation and charge recombination processes in Synthetic DNA hairpins have been investigated by means of femtosecond transient absorption spectroscopy. The driving force and distance dependence of charge-transfer processes involving singlet acceptors and nucleobase donors are consistent with a single-step superexchange mechanism in which the electronic coupling between the donor and acceptor is strongly distance dependent. The dynamics of reversible hole transport between a primary guanine donor and nearby GG or GGG sequences has also been determined and establishes that these sequences are very shallow hole traps.

  • Dynamics of Photoinduced Charge Transfer and Hole Transport in Synthetic DNA
    2001
    Co-Authors: Robert L Letsinger, Michael R Wasielewski
    Abstract:

    The dynamics of photoinduced charge separation and charge recombination processes in Synthetic DNA hairpins have been investigated by means of femtosecond transient absorption spectroscopy. The driving force and distance dependence of chargetransfer processes involving singlet acceptors and nucleobase donors are consistent with a single-step superexchange mechanism in which the electronic coupling between the donor and acceptor is strongly distance dependent. The dynamics of reversible hole transport between a primary guanine donor and nearby GG or GGG sequences has also been determined and establishes that these sequences are very shallow hole traps.

  • Driving force dependence of electron transfer dynamics in Synthetic DNA hairpins
    Journal of the American Chemical Society, 2000
    Co-Authors: Frederick D Lewis, Rajdeep S. Kalgutkar, Xiaoyang Liu, Jianqin Liu, Ryan T. Hayes, And Scott E. Miller, Michael R Wasielewski
    Abstract:

    The driving force dependence of photoinduced electron-transfer dynamics in duplex DNA has been investigated for 16 Synthetic DNA hairpins in which an acceptor chromophore serves as a linker connecting two complementary oligonucleotide arms containing a single donor nucleobase located either adjacent to the linker or separated from the linker by two unreactive base pairs. The rate constants for both charge separation and charge recombination processes have been determined by means of subpicosecond time-resolved transient absorption spectroscopy and the results analyzed using quantum mechanical Marcus theory. This analysis provides intimate details about electron-transfer processes in DNA including the distance dependence of the electronic coupling between the acceptor and nucleobase donor and the solvent and nuclear reorganization energies.

  • Dynamics of Photoinduced Charge Separation and Charge Recombination in Synthetic DNA Hairpins with Stilbenedicarboxamide Linkers
    Journal of the American Chemical Society, 2000
    Co-Authors: Frederick D Lewis, Robert L Letsinger, Xiaoyang Liu, Scott E. Miller, Scott R. Greenfield, Michael R Wasielewski
    Abstract:

    The dynamics of photoinduced charge separation and charge recombination in Synthetic DNA hairpins have been investigated by means of femtosecond and nanosecond transient spectroscopy. The hairpins consist of a stilbene linker connecting two complementary 6-mer or 7-mer oligonucleotide strands. Base pairing between these strands results in formation of hairpins in which the stilbene is approximately parallel to the adjacent base pair. The singlet stilbene is selectively quenched by guanine, but not by the other nucleobases, via an electron-transfer mechanism in which the stilbene singlet state is the electron acceptor and guanine is the electron donor. In a hairpin containing only A:T base pairs, no quenching occurs and the restricted geometry results in a long stilbene lifetime and high fluorescence quantum yield. In families of hairpins which contain a single G:C base pair at varying locations in the hairpin stem, the stilbene fluorescence lifetime and quantum yield decrease as the stilbene−guanine dista...

  • Structure and photoinduced electron transfer in exceptionally stable Synthetic DNA hairpins with stilbenediether linkers
    Journal of the American Chemical Society, 1999
    Co-Authors: Frederick D Lewis, Michael R Wasielewski, Robert L Letsinger, Xiaoyang Liu, Scott E. Miller, Ruslan Sanishvili, Andrzej Joachimiak, Valentina Tereshko, Martin Egli
    Abstract:

    Bis(oligonucleotide) conjugates with Synthetic linkers connecting short complementary oligonucleotides are known to form Synthetic DNA or RNA hairpins which are, in some cases, more stable than natural hairpins which possess oligonucleotide linkers. The authors report here that conjugates possessing stilbenediether (SE) linkers form exceptionally stable (poly)dT-SE-(poly)dA hairpins. The crystal structure of a Se-bridged hairpin confirms that it adopts a B-form structure in which the stilbene is {pi}-stacked with the adjacent base pair. The stilbenediether also mediates novel lattice interactions that are distinct from those normally found in DNA crystals. The singlet excited state of the stilbenediether is a strong electron donor which is rapidly quenched by either neighboring dT-dA or dC-dG base pairs which function as electron acceptors. This behavior is complementary to that of conjugates possessing a stilbenedicarboxamide linker (SA, Chart 1), which serves as an electron acceptor.

Frederick D Lewis - One of the best experts on this subject based on the ideXlab platform.

  • dynamics of photoinduced charge transfer and hole transport in Synthetic DNA hairpins
    Accounts of Chemical Research, 2001
    Co-Authors: Frederick D Lewis, Robert L Letsinger, Michael R Wasielewski
    Abstract:

    The dynamics of photoinduced charge separation and charge recombination processes in Synthetic DNA hairpins have been investigated by means of femtosecond transient absorption spectroscopy. The driving force and distance dependence of charge-transfer processes involving singlet acceptors and nucleobase donors are consistent with a single-step superexchange mechanism in which the electronic coupling between the donor and acceptor is strongly distance dependent. The dynamics of reversible hole transport between a primary guanine donor and nearby GG or GGG sequences has also been determined and establishes that these sequences are very shallow hole traps.

  • Driving force dependence of electron transfer dynamics in Synthetic DNA hairpins
    Journal of the American Chemical Society, 2000
    Co-Authors: Frederick D Lewis, Rajdeep S. Kalgutkar, Xiaoyang Liu, Jianqin Liu, Ryan T. Hayes, And Scott E. Miller, Michael R Wasielewski
    Abstract:

    The driving force dependence of photoinduced electron-transfer dynamics in duplex DNA has been investigated for 16 Synthetic DNA hairpins in which an acceptor chromophore serves as a linker connecting two complementary oligonucleotide arms containing a single donor nucleobase located either adjacent to the linker or separated from the linker by two unreactive base pairs. The rate constants for both charge separation and charge recombination processes have been determined by means of subpicosecond time-resolved transient absorption spectroscopy and the results analyzed using quantum mechanical Marcus theory. This analysis provides intimate details about electron-transfer processes in DNA including the distance dependence of the electronic coupling between the acceptor and nucleobase donor and the solvent and nuclear reorganization energies.

  • Dynamics of Photoinduced Charge Separation and Charge Recombination in Synthetic DNA Hairpins with Stilbenedicarboxamide Linkers
    Journal of the American Chemical Society, 2000
    Co-Authors: Frederick D Lewis, Robert L Letsinger, Xiaoyang Liu, Scott E. Miller, Scott R. Greenfield, Michael R Wasielewski
    Abstract:

    The dynamics of photoinduced charge separation and charge recombination in Synthetic DNA hairpins have been investigated by means of femtosecond and nanosecond transient spectroscopy. The hairpins consist of a stilbene linker connecting two complementary 6-mer or 7-mer oligonucleotide strands. Base pairing between these strands results in formation of hairpins in which the stilbene is approximately parallel to the adjacent base pair. The singlet stilbene is selectively quenched by guanine, but not by the other nucleobases, via an electron-transfer mechanism in which the stilbene singlet state is the electron acceptor and guanine is the electron donor. In a hairpin containing only A:T base pairs, no quenching occurs and the restricted geometry results in a long stilbene lifetime and high fluorescence quantum yield. In families of hairpins which contain a single G:C base pair at varying locations in the hairpin stem, the stilbene fluorescence lifetime and quantum yield decrease as the stilbene−guanine dista...

  • Structure and photoinduced electron transfer in exceptionally stable Synthetic DNA hairpins with stilbenediether linkers
    Journal of the American Chemical Society, 1999
    Co-Authors: Frederick D Lewis, Michael R Wasielewski, Robert L Letsinger, Xiaoyang Liu, Scott E. Miller, Ruslan Sanishvili, Andrzej Joachimiak, Valentina Tereshko, Martin Egli
    Abstract:

    Bis(oligonucleotide) conjugates with Synthetic linkers connecting short complementary oligonucleotides are known to form Synthetic DNA or RNA hairpins which are, in some cases, more stable than natural hairpins which possess oligonucleotide linkers. The authors report here that conjugates possessing stilbenediether (SE) linkers form exceptionally stable (poly)dT-SE-(poly)dA hairpins. The crystal structure of a Se-bridged hairpin confirms that it adopts a B-form structure in which the stilbene is {pi}-stacked with the adjacent base pair. The stilbenediether also mediates novel lattice interactions that are distinct from those normally found in DNA crystals. The singlet excited state of the stilbenediether is a strong electron donor which is rapidly quenched by either neighboring dT-dA or dC-dG base pairs which function as electron acceptors. This behavior is complementary to that of conjugates possessing a stilbenedicarboxamide linker (SA, Chart 1), which serves as an electron acceptor.

Tim R. Mercer - One of the best experts on this subject based on the ideXlab platform.

  • A universal and independent Synthetic DNA ladder for the quantitative measurement of genomic features.
    Nature communications, 2020
    Co-Authors: Andre L. M. Reis, Ira W. Deveson, Ted Wong, James Blackburn, Bindu Swapna Madala, Chris Barker, Esteban Marcellin, Tim R. Mercer
    Abstract:

    Standard units of measurement are required for the quantitative description of nature; however, few standard units have been established for genomics to date. Here, we have developed a Synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA sample, and act as an internal scale that measures quantitative genetics features. Unlike previous spike-ins, the ladder is encoded within a single molecule, and can be equivalently and independently synthesized by different laboratories. We show how the ladder can measure diverse quantitative features, including human genetic variation and microbial abundance, and also estimate uncertainty due to technical variation and improve normalization between libraries. This ladder provides an independent quantitative unit that can be used with any organism, application or technology, thereby providing a common metric by which genomes can be measured.

  • Use of Synthetic DNA spike-in controls (sequins) for human genome sequencing
    Nature Protocols, 2019
    Co-Authors: James Blackburn, Ira W. Deveson, Ted Wong, Simon A. Hardwick, Bindu Swapna Madala, Chris Barker, Andre L. M. Reis, Tim R. Mercer
    Abstract:

    Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of Synthetic DNA spike-ins—termed ‘sequins’ (sequencing spike-ins)—that are directly added to DNA samples before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA samples, followed by the bioinformatic steps required to separate sequin- and sample-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1–4 d and the bioinformatic steps (which can be performed with the provided example data files) take an additional day. Here, the authors describe the use of Synthetic DNA spike-ins, called sequins, as internal quantitative and qualitative controls throughout the sequencing workflow. They detail their application in whole-genome and targeted human genome sequencing.

  • Use of Synthetic DNA spike-in controls (sequins) for human genome sequencing.
    Nature protocols, 2019
    Co-Authors: James Blackburn, Ira W. Deveson, Ted Wong, Simon A. Hardwick, Bindu Swapna Madala, Chris Barker, Andre L. M. Reis, Tim R. Mercer
    Abstract:

    Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of Synthetic DNA spike-ins-termed 'sequins' (sequencing spike-ins)-that are directly added to DNA samples before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA samples, followed by the bioinformatic steps required to separate sequin- and sample-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1-4 d and the bioinformatic steps (which can be performed with the provided example data files) take an additional day.

  • Representing genetic variation with Synthetic DNA standards
    Nature methods, 2016
    Co-Authors: Ira W. Deveson, Wendy Y. Chen, Ted Wong, Simon A. Hardwick, Stacey B. Andersen, Lars K. Nielsen, John S. Mattick, Tim R. Mercer
    Abstract:

    The identification of genetic variation with next-generation sequencing is confounded by the complexity of the human genome sequence and by biases that arise during library preparation, sequencing and analysis. We have developed a set of Synthetic DNA standards, termed 'sequins', that emulate human genetic features and constitute qualitative and quantitative spike-in controls for genome sequencing. Sequencing reads derived from sequins align exclusively to an artificial in silico reference chromosome, rather than the human reference genome, which allows them them to be partitioned for parallel analysis. Here we use this approach to represent common and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements and copy-number variation. We validate the design and performance of sequin standards by comparison to examples in the NA12878 reference genome, and we demonstrate their utility during the detection and quantification of variants. We provide sequins as a standardized, quantitative resource against which human genetic variation can be measured and diagnostic performance assessed.

Nadrian C. Seeman - One of the best experts on this subject based on the ideXlab platform.

  • Topological transformations of Synthetic DNA knots.
    Biochemistry, 1995
    Co-Authors: Hui Wang, Yuk-ching Tse-dinh, Nadrian C. Seeman
    Abstract:

    Two Synthetic DNA molecules that can be knotted have been employed as substrates for E. coli DNA topoisomerases I and III. Both molecules contain 104 nucleotides, including sequences that can form two single-turn helical domains, connected by single-stranded oligo(dT) linkers in an X-Y-X'-Y' pairing motif. One of the knots can be ligated to form cyclic molecules with the topologies of a circle, a trefoil knot with negative nodes, or a figure-8 knot. Cyclic molecules constructed from the other molecule can form a circle, a figure-8 knot, and trefoil knots with either positive or negative nodes. The topologically negative nodes in these knots are derived from right-handed B-DNA, and the positive nodes are derived from left-handed Z-DNA. The topoisomerases can catalyze the interconversion of the different topological forms of these molecules, as a function of solution conditions and the extent to which they favor B-DNA or Z-DNA. The enzymes appear to catalyze a single strand-passage event at a time. The topoisomerases can catalyze strand passage events involving both positive and negative nodes as substrates. Gel retention experiments show that both knots can bind up to four molecules of E. coli DNA topoisomerase I. The thermal denaturation of the domains of a trefoil knot closely related to these knots suggests that the two helical domains are uncoupled, so the single-stranded linkers in the knots are not taut. Chemical ligation experiments yield a distribution of products similar to those of enzymatic ligation, showing that the ATP cofactor in DNA knot ligation does not appear to skew the products markedly. Knots that are stressed by being placed in unfavorable solution conditions have been shown to be a highly sensitive system for detecting topoisomerase activity.

  • Synthetic DNA knots and catenanes
    New Journal of Chemistry, 1993
    Co-Authors: Nadrian C. Seeman, Hui Wang, Junghuei Chen, Shou Ming Du, John E. Mueller, Yuqi Zhang, Tsu-ju Fu, Yi Lu Wang, Siwei Zhang
    Abstract:

    The double helical structure of DNA can be used as the basis for the synthesis of specific single-stranded catenanes and knots. It is possible to construct DNA stick figures, such as a cube, from branched DNA components. The edges of these molecules are double helical DNA, and the individual strands combine to form complex DNA catenanes. The sequences of these molecules are selected by minimizing the sequence symmetry of the component strands, and they are ligated together by techniques used routinely in biotechnology. Precise catenation is controlled by the use of topological protecting groups, to prevent braiding that is not wanted in the target molecule. Solid-support based assembly techniques have been developed to facilitate the construction of these objects