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

Modern Approaches to de novo Synthesis of Extended DNA Fragments: Assembly of a Wide Repertoire of Sequences

“…The main goals are to obtain longer sequences (more than 100 bp), increase the yield of the reaction at all stages of the synthetic cycle, and reduce the number of errors by improving the quality of chemical reagents [ 12 ]. Three main strategies for the assembly of oligonucleotides into dsDNA fragments have been developed: in vitro assembly using enzymes – ligase cyclic assembly (LCR) [ 13 , 14 ] and polymerase cyclic assembly (PCR), as well as in vivo assembly in yeast cells [ 15 ]. The main advantages of PCR assembly are the smaller concentration of the oligonucleotides required for the reaction, the absence of an oligonucleotide phosphorylation stage, and lower labor intensity [ 16 , 17 ].…”

“…), увеличение выхода реакции на всех этапах синтетического цикла, снижение количества ошибок за счет улучше-ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ ния качества химических реагентов [12]. Разработаны три основных стратегии сборки олигонуклеотидов в дцДНК-фрагменты -сборка in vitro с помощью ферментов -лигазная циклическая сборка (ЛЦР) [13,14], полимеразная циклическая сборка (ПЦР), а также in vivo сборка в клетках дрожжей [15]. Основными преимуществами сборки с помощью ПЦР являются меньшее количество олигонуклеотидов, необходимое для проведения реакции, отсутствие стадии фосфорилирования олигонуклеотидов и меньшая трудоемкость [16,17].…”

“In vitro construction and long read sequencing analysis of a 24 kb long artificial DNA sequence encoding the Universal Declaration of the Rights of Man and of the Citizen ”

“…Many methods have been developed to construct large DNA fragments from short chemically synthesized DNA oligonucleotides ( 6,15,24 ). The most common are Golden Gate assembly (GGA), Gibson cloning and polymerase cycling assembly.…”