Programmed assembly of long DNA synthons: design, mechanism, and online monitoring
- 46 Downloads
Synthesis of custom de novo DNA sequences is highly demanded by fast-growing field of synthetic biology. Usually DNA sequences with length more than 1 kb are assembled from smaller synthetic DNA fragments (synthons) obtained by PCR assembly. The ability to synthesize longer synthons sufficiently reduces efforts and time for DNA synthesis. We developed a novel rational oligonucleotide design and programmed approach for the assembly of synthetic DNA synthons up to 1550 bp. The developed procedure was thoroughly investigated by synthesis of cholesterol oxidase gene from Streptomyces lavendulae (1544 bp). Our approach is based on combined design, oligonucleotide concentration gradient, and specialized assembly program that directs assembly reaction to full-length gene in a stepwise manner. The process includes conventional thermodynamically balanced assembly, thermodynamically balanced inside-out elongation, and further amplification. The ability of DNA polymerase to perform programmed assembly is highly influenced by the presence of 5′ → 3′-exonuclease activity. Oligonucleotide probing of PCR assembly products allowed us to shed light on the nature of high molecular weight spurious by-products and to understand the mechanism of their formation. For the first time, we applied light scattering techniques for tracking of oligonucleotide annealing, analysis of gene assembly products, and even for real-time monitoring of gene assembly process.
KeywordsGene synthesis Concatemer Dynamic light scattering Polymerase chain reaction Cholesterol oxidase
This work is funded by Separate Project from National Academy of Sciences of Belarus.
Compliance with ethical standards
Research involving human participants and/or animals
The authors declare that that work did not require human and/or animal participation.
Conflict of interest
The authors declare that they have no conflict of interest.
- Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361. https://doi.org/10.1016/B978-0-12-385120-8.00015-2 CrossRefGoogle Scholar
- Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, Smith HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215–1220. https://doi.org/10.1126/science.1151721 CrossRefGoogle Scholar
- Hughes RA, Miklos AE, Ellington AD (2011) Gene synthesis: methods and applications. Methods Enzymol 498:277–309. https://doi.org/10.1016/B978-0-12-385120-8.00012-7 CrossRefGoogle Scholar
- Strizhov N, Keller M, Mathur J, Koncz-Kalman Z, Bosch D, Prudovsky E, Schell J, Sneh B, Koncz C, Zilberstein A (1996) A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci U S A 93(26):15012–15017CrossRefGoogle Scholar