The Experts below are selected from a list of 212334 Experts worldwide ranked by ideXlab platform
Daniel V Santi - One of the best experts on this subject based on the ideXlab platform.
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gems an advanced software package for designing synthetic Genes
Nucleic Acids Research, 2005Co-Authors: Sebastian Jayaraj, Ralph Reid, Daniel V SantiAbstract:A user-friendly, advanced software package for Gene design is described. The software comprises an integrated suite of programs-also provided as stand-alone tools-that automatically performs the following tasks in Gene design: restriction site prediction, codon optimization for any expression host, restriction site inclusion and exclusion, separation of long sequences into synthesizable fragments, T(m) and stem-loop determinations, optimal oligonucleotide component design and design verification/error-checking. The output is a complete design report and a list of optimized oligonucleotides to be prepared for subsequent Gene Synthesis. The user interface accommodates both inexperienced and experienced users. For inexperienced users, explanatory notes are provided such that detailed instructions are not necessary; for experienced users, a streamlined interface is provided without such notes. The software has been extensively tested in the design and successful Synthesis of over 400 kb of Genes, many of which exceeded 5 kb in length.
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total Synthesis of long dna sequences Synthesis of a contiguous 32 kb polyketide synthase Gene cluster
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sarah J Kodumal, Ralph Reid, Kedar G Patel, Hugo G Menzella, Mark Welch, Daniel V SantiAbstract:To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the Synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440–15445]. The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput Synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based Gene Synthesis. By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection.” These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase Gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.
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total Synthesis of long dna sequences Synthesis of a contiguous 32 kb polyketide synthase Gene cluster
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sarah J Kodumal, Ralph Reid, Kedar G Patel, Hugo G Menzella, Mark Welch, Daniel V SantiAbstract:To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the Synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440–15445]. The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput Synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based Gene Synthesis. By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection.” These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase Gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.
Jingdong Tian - One of the best experts on this subject based on the ideXlab platform.
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dna Synthesis assembly and applications in synthetic biology
Current Opinion in Chemical Biology, 2012Co-Authors: Nicholas Tang, Jingdong TianAbstract:The past couple of years saw exciting new developments in microchip-based Gene Synthesis technologies. Such technologies hold the potential for significantly increasing the throughput and decreasing the cost of Gene Synthesis. Together with more efficient enzymatic error correction and genome assembly methods, these new technologies are pushing the field of synthetic biology to a higher level.
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error correction of microchip synthesized Genes using surveyor nuclease
Nucleic Acids Research, 2012Co-Authors: Ishtiaq Saaem, Jiayuan Quan, Jingdong TianAbstract:The development of economical and highthroughput Gene Synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic Genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic Gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3 0 !5 0 exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact Genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic Genes by >16-fold, yielding a final error rate of � 1 in 8700bp.
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parallel on chip Gene Synthesis and application to optimization of protein expression
Nature Biotechnology, 2011Co-Authors: Jiayuan Quan, Hui Gong, Ishtiaq Saaem, Nicholas Tang, Nicolas Negre, Kevin P White, Jingdong TianAbstract:Low-cost, high-throughput Gene Synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip Gene Synthesis technology, which integrates on a single microchip the Synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel Gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of Synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.
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parallel on chip Gene Synthesis and application to optimization of protein expression
Nature Biotechnology, 2011Co-Authors: Jiayuan Quan, Hui Gong, Ishtiaq Saaem, Nicholas Tang, Nicolas Negre, Kevin P White, Jingdong TianAbstract:High-throughput Synthesis of long DNA molecules would open up new experimental paradigms in synthetic biology and functional genomics. Quan et al. take a step toward this goal by integrating oligonucleotide Synthesis, amplification and Gene assembly on a single microarray, and apply the technology to optimization of protein translation in a heterologous host.
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accurate multiplex Gene Synthesis from programmable dna microchips
Nature, 2004Co-Authors: Jingdong Tian, Hui Gong, Nijing Sheng, Xiaochuan Zhou, Erdogan Gulari, Xiaolian Gao, George M ChurchAbstract:Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective Gene and genome Synthesis. Here we describe a microchip-based technology for multiplex Gene Synthesis. Pools of thousands of ‘construction’ oligonucleotides and tagged complementary ‘selection’ oligonucleotides are synthesized on photo-programmable microfluidic chips1, released, amplified and selected by hybridization to reduce Synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple Genes. This technology enabled us to synthesize all 21 Genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the Synthesis of ribosomes in vitro and should have utility for synthetic biology in General.
George M Church - One of the best experts on this subject based on the ideXlab platform.
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a systematic comparison of error correction enzymes by next Generation sequencing
Nucleic Acids Research, 2017Co-Authors: Nathan B. Lubock, Angus M. Sidore, Di Zhang, George M Church, Sriram KosuriAbstract:Gene Synthesis, the process of assembling Gene-length fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of Gene Synthesis are limited by errors produced during oligo Synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in Gene Synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-Generation sequencing. We analyzed errors in model Gene assemblies and systematically compared six different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More Generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in Gene assemblies, to profile error correction methods, and to benchmark DNA Synthesis methods.
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a systematic comparison of error correction enzymes by next Generation sequencing
bioRxiv, 2017Co-Authors: Nathan B. Lubock, Di Zhang, George M Church, Sriram KosuriAbstract:Gene Synthesis, the process of assembling Gene-length fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality, and cost of Gene Synthesis is limited by errors produced during oligo Synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in Gene Synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment and throughput. Here we develop a method to quantify errors in synthetic DNA by next-Generation sequencing. We analyzed errors in a model Gene assembly and systematically compared six different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G → G/C transversions whereas T7 Endonuclease I preferentially corrects A/T → T/A transversions. More Generally, this experimental and computational pipeline is a fast, scalable, and extensible way to analyze errors in Gene assemblies, to profile error correction methods, and to benchmark DNA Synthesis methods.
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iterative capped assembly rapid and scalable Synthesis of repeat module dna such as tal effectors from individual monomers
Nucleic Acids Research, 2012Co-Authors: Adrian W Briggs, Xavier Rios, Raj Chari, Luhan Yang, Feng Zhang, Prashant Mali, George M ChurchAbstract:DNA built from modular repeats presents a challenge for Gene Synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA toamodelproblem,constructionofcustomtranscription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient Synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nucleasecarrying backbone vector all within 3h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate Gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical.
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scalable Gene Synthesis by selective amplification of dna pools from high fidelity microchips
Nature Biotechnology, 2010Co-Authors: Sriram Kosuri, George M Church, Nikolai Eroshenko, Emily M Leproust, Michael Super, Jeffrey C WayAbstract:Development of cheap, high-throughput and reliable Gene Synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in Gene Synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized Gene assembly protocols and enzymatic error correction to develop a method for highly parallel Gene Synthesis. We tested our approach by assembling 47 Genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.
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accurate multiplex Gene Synthesis from programmable dna microchips
Nature, 2004Co-Authors: Jingdong Tian, Hui Gong, Nijing Sheng, Xiaochuan Zhou, Erdogan Gulari, Xiaolian Gao, George M ChurchAbstract:Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective Gene and genome Synthesis. Here we describe a microchip-based technology for multiplex Gene Synthesis. Pools of thousands of ‘construction’ oligonucleotides and tagged complementary ‘selection’ oligonucleotides are synthesized on photo-programmable microfluidic chips1, released, amplified and selected by hybridization to reduce Synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple Genes. This technology enabled us to synthesize all 21 Genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the Synthesis of ribosomes in vitro and should have utility for synthetic biology in General.
Kedar G Patel - One of the best experts on this subject based on the ideXlab platform.
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high throughput cost effective verification of structural dna assembly
Nucleic Acids Research, 2014Co-Authors: Yandi Dharmadi, Kedar G Patel, Elaine Shapland, Daniel Hollis, Todd Slaby, Nicole Klinkner, Jed Dean, Sunil S ChandranAbstract:DNA 'assembly' from 'building blocks' remains a cornerstone in synthetic biology, whether it be for Gene Synthesis (∼ 1 kb), pathway engineering (∼ 10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.
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total Synthesis of long dna sequences Synthesis of a contiguous 32 kb polyketide synthase Gene cluster
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sarah J Kodumal, Ralph Reid, Kedar G Patel, Hugo G Menzella, Mark Welch, Daniel V SantiAbstract:To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the Synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440–15445]. The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput Synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based Gene Synthesis. By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection.” These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase Gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.
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total Synthesis of long dna sequences Synthesis of a contiguous 32 kb polyketide synthase Gene cluster
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sarah J Kodumal, Ralph Reid, Kedar G Patel, Hugo G Menzella, Mark Welch, Daniel V SantiAbstract:To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the Synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440–15445]. The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput Synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based Gene Synthesis. By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection.” These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase Gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.
Ralph Reid - One of the best experts on this subject based on the ideXlab platform.
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gems an advanced software package for designing synthetic Genes
Nucleic Acids Research, 2005Co-Authors: Sebastian Jayaraj, Ralph Reid, Daniel V SantiAbstract:A user-friendly, advanced software package for Gene design is described. The software comprises an integrated suite of programs-also provided as stand-alone tools-that automatically performs the following tasks in Gene design: restriction site prediction, codon optimization for any expression host, restriction site inclusion and exclusion, separation of long sequences into synthesizable fragments, T(m) and stem-loop determinations, optimal oligonucleotide component design and design verification/error-checking. The output is a complete design report and a list of optimized oligonucleotides to be prepared for subsequent Gene Synthesis. The user interface accommodates both inexperienced and experienced users. For inexperienced users, explanatory notes are provided such that detailed instructions are not necessary; for experienced users, a streamlined interface is provided without such notes. The software has been extensively tested in the design and successful Synthesis of over 400 kb of Genes, many of which exceeded 5 kb in length.
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total Synthesis of long dna sequences Synthesis of a contiguous 32 kb polyketide synthase Gene cluster
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sarah J Kodumal, Ralph Reid, Kedar G Patel, Hugo G Menzella, Mark Welch, Daniel V SantiAbstract:To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the Synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440–15445]. The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput Synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based Gene Synthesis. By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection.” These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase Gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.
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total Synthesis of long dna sequences Synthesis of a contiguous 32 kb polyketide synthase Gene cluster
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sarah J Kodumal, Ralph Reid, Kedar G Patel, Hugo G Menzella, Mark Welch, Daniel V SantiAbstract:To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the Synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440–15445]. The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput Synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based Gene Synthesis. By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection.” These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase Gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.