Simplified gene synthesis: A one-step approach to PCR-based gene construction

Wu, Gang; Wolf, Julie B.; Ibrahim, Ameer F.; Vadasz, Stephanie; Gunasinghe, Muditha; Freeland, Stephen J.

doi:10.1016/j.jbiotec.2006.01.015

Cited by 52 publications

(62 citation statements)

References 21 publications

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“…It is possible, however that errors cluster near short component oligos with lower annealing temperatures as may be the case for the oligo pair containing the exchangeable exposed site. The preponderance of deletions in gene synthesis errors could be attributed to the use of a proofreading polymerase, as has previously been reported (23).…”

Section: In Vivo Functionality Of Synthetic Promoterssupporting

confidence: 61%

Rapid Synthesis of Defined Eukaryotic Promoter Libraries

Rajkumar

Maerkl

2012

ACS Synth. Biol.

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Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. Overall, 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions and may serve as tunable components for synthetic genetic networks.

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Section: In Vivo Functionality Of Synthetic Promoterssupporting

confidence: 61%

Rapid Synthesis of Defined Eukaryotic Promoter Libraries

Rajkumar

Maerkl

2012

ACS Synth. Biol.

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“…To generate the NKX3.1 KO mutant, a codon-optimized gene was generated by simplified gene synthesis in which the 10 homeodomain lysines were mutated by using multiple primers listed in supplemental data (28). The remaining four lysines were mutated by QuikChange site-directed mutagenesis using primers described in supplemental data.…”

Section: Methodsmentioning

confidence: 99%

Proline-mediated Proteasomal Degradation of the Prostate-specific Tumor Suppressor NKX3.1

Rao

Guan

Mutton

et al. 2012

Journal of Biological Chemistry

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Background:The prostate-specific tumor suppressor NKX3.1 is targeted for proteasomal degradation. Results: A proline-dependent C-terminal 21 amino acid portable degron regulates NKX3.1 ubiquitin-independent degradation in prostate cancer cells. Conclusion: NKX3.1 proteasomal degradation is mediated by both ubiquitin-dependent and -independent pathways. Significance: Understanding mechanisms regulating NKX3.1 turnover provides opportunities to develop tumor suppressor restoration therapies in prostate cancer.

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“…Factors that were tested include choice of DNA polymerase and buffer, total and individual oligonucleotide concentrations, oligonucleotide purity, annealing temperature, and number of thermocycling steps (Electronic Supplemental Material). We, and others, have found that the DNA polymerase choice is a critical determinant for robust assembly (Wu et al 2006), with KOD polymerase derived from Thermococcus kodakaraensis being the most successful (Takagi et al 1997). Use of a concentration gradient for the ION pair oligonucleotides is also critical.…”

Section: Automation Schemementioning

confidence: 99%

Protein fabrication automation

et al. 2007

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Facile ''writing'' of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.

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Simplified gene synthesis: A one-step approach to PCR-based gene construction

Cited by 52 publications

References 21 publications

Rapid Synthesis of Defined Eukaryotic Promoter Libraries

Rapid Synthesis of Defined Eukaryotic Promoter Libraries

Proline-mediated Proteasomal Degradation of the Prostate-specific Tumor Suppressor NKX3.1

Protein fabrication automation

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