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Applications of microchip-synthesized oligonucleotides for de novo gene synthesis are limited primarily by their high error rates. The mismatch binding protein MutS, which can specifically recognize and bind to mismatches, is one of the cheapest tools for error correction of synthetic DNA. Here, we describe a protocol for removing errors in microchip-synthesized oligonucleotides and for the assembly of DNA segments using these oligonucleotides. This protocol can also be used in traditional de novo gene DNA synthesis.
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http://dx.doi.org/10.1007/978-1-4939-6343-0_17 | DOI Listing |
Sci Rep
July 2017
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China.
Engineering and evaluation of synthetic routes for generating valuable compounds require accurate and cost-effective de novo synthesis of genetic pathways. Here, we present an economical and streamlined de novo DNA synthesis approach for engineering a synthetic pathway with microchip-synthesized oligonucleotides (oligo). The process integrates entire oligo pool amplification, error-removal, and assembly of long DNA molecules.
View Article and Find Full Text PDFNanoscale
March 2017
Department of Biomedical Engineering, Duke University, Durham, 27705, USA. and Department of Biomedical Engineering, Columbia University, New York, 10027, USA and Department of Systems Biology, Columbia University, New York, 10027, USA.
The rapid advances in synthetic biology and biotechnology are increasingly demanding high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase.
View Article and Find Full Text PDFMethods Mol Biol
January 2018
School of Life Science, University of Science and Technology of China, No 443 Huangshan Road, Hefei, Anhui, 230026, People's Republic of China.
Applications of microchip-synthesized oligonucleotides for de novo gene synthesis are limited primarily by their high error rates. The mismatch binding protein MutS, which can specifically recognize and bind to mismatches, is one of the cheapest tools for error correction of synthetic DNA. Here, we describe a protocol for removing errors in microchip-synthesized oligonucleotides and for the assembly of DNA segments using these oligonucleotides.
View Article and Find Full Text PDFJ Biotechnol
January 2015
School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China. Electronic address:
We report a rational strategy to design and construct multiple small perturbation mutagenesis (SPM) libraries using massively parallel synthesis of oligonucleotides on a microchip for affinity maturation of an engineered anti-ErbB2 antibody chA21. On the basis of a comprehensive analysis of the sequence and structural relationships of six complementary determination regions (CDRs) in the Kabatman database, a computational algorithm was developed to introduce single-site and double-site mutations into variable CDR positions using ambiguous nucleotides. The six SPM libraries were composed of 419 degenerate oligonucleotides that can be expanded into 161,832 unique CDR sequences with a high coverage ratio of 95% natural amino acid diversity.
View Article and Find Full Text PDFNucleic Acids Res
July 2014
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China
The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly.
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