Modern approaches to artificial gene synthesis: aspects of oligonucleotide synthesis, enzymatic assembly, sequence verification and error correction

G Y Shevelev,D V Pyshnyi

doi:10.18699/vj18.387

Abstract

Synthetic biology is a rapidly developing field aimed at engineering of biological systems with predictable properties. Synthetic biology accumulates the achievements of modern biological sciences, programming and computational modeling as well as engineering technologies for creation of biological objects with user-defined properties. Evolution of synthetic biology has been marked by a number of technological developments in each of the mentioned fields. Thus, significant reduction in cost of DNA sequencing has provided an easy access to large amounts of data on the genetic sequences of various organisms, and decreased the price of the DNA sequence synthesis, which, analogous to Moore’s law, resulted in an opportunity to create a lot of potential genes without the time – consuming and labor – intensive traditional methods of molecular biology. Development of system biology has allowed forming a deeper understanding of the functions and relationship of natural biological models, as well as of the computational models describing processes at the cell and system levels. Combination of these factors has created an opportunity for conscious changes of natural biological systems. In this review the modern approaches to oligonucleotide gene assembly synthesis are discussed, including such aspects as protocols for gene assembly, sequence verification, error correction and further applications of synthesized genes.

Highlights

Синтетическая биология – быстро развивающаяся отрасль науки, нацеленная на создание биологических систем с предсказанными свойствами
It relies on the modern methods of oligonucleotide synthesis, the variants of Polymerase Chain Reaction (PCR) (Saiki et al, 1985; Mullis et al, 1986) with high fidelity DNA polymerases (Böhlke et al, 2000), and the sequence verification step using the Sanger sequencing (Sanger, Coulson, 1975; Smith et al, 1986) or high-throughput DNA sequencing (Church, 2006; Hall, 2007; Bosch, Grody, 2008; Schuster, 2008; Shendure, Ji, 2008; Tucker et al, 2009)
The DNA oligonucleotides are synthesized from the 3′ to 5′ end by consecutive coupling of activated building deoxynucleoside phosphoramidites to an initial deoxynucleoside attached to a solid support (usually the support is a controlled pore glass (CPG) or highly cross-linked polystyrene beads) by its 3′-OH group (Ellington, Pollard, 2000)

Summary

Молекулярная и клеточная биология

Modern approaches to artificial gene synthesis: aspects of oligonucleotide synthesis, enzymatic assembly, sequence verification and error correction. Since a first gene was synthesized from scratch in the 70’s (Agarwal et al, 1970) and it’s in vivo activity was tested (Ryan et al, 1979) around 50 years has passed, and the technology of artificial gene synthesis has made a significant leap beyond the approaches implemented in the classical works Nowdays, it relies on the modern methods of oligonucleotide synthesis, the variants of Polymerase Chain Reaction (PCR) (Saiki et al, 1985; Mullis et al, 1986) with high fidelity DNA polymerases (Böhlke et al, 2000), and the sequence verification step using the Sanger sequencing (Sanger, Coulson, 1975; Smith et al, 1986) or high-throughput DNA sequencing (Church, 2006; Hall, 2007; Bosch, Grody, 2008; Schuster, 2008; Shendure, Ji, 2008; Tucker et al, 2009). Modern approaches to artificial gene synthesis: oligo synthesis, enzymatic assembly, sequence verification and error correction

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Findings

Molecular and cell biology