Overview of Post Cohen‐Boyer Methods for Single Segment Cloning and for Multisegment DNA Assembly

Abstract

In 1973, Cohen and coworkers published a foundational paper describing the cloning of DNA fragments into plasmid vectors. In it, they used DNA segments made by digestion with restriction enzymes and joined these in vitro with DNA ligase. These methods established working recombinant DNA technology and enabled the immediate start of the biotechnology industry. Since then, “classical” recombinant DNA technology using restriction enzymes and DNA ligase has matured. At the same time, researchers have developed numerous ways to generate large, complex, multisegment DNA constructions that offer advantages over classical techniques. Here, we provide an overview of “post-Cohen-Boyer” techniques used for cloning single segments into vectors (T/A, Topo cloning, Gateway and Recombineering) and for multisegment DNA assembly (Biobricks, Golden Gate, Gibson, Yeast homologous recombination in vivo, and Ligase Cycling Reaction). We compare and contrast these methods and also discuss issues that researchers should consider before choosing a particular multisegment DNA assembly method.

Expression and Purification of Recombinant Proteins Using the Baculovirus System

“…To circumvent this problem, a number of plasmid systems have been developed for expressing multisubunit complexes from a single baculovirus, including the biGBac system by Weissmann et al (2016). Cloning in this system relies on one-step isothermal DNA assembly [Gibson et al 2009; also see Sands and Brent (2016) and Yang et al (2016)] to generate a multigene expression plasmid containing up to 25 genes of interest.…”

Using measures of single‐cell physiology and physiological state to understand organismic aging

“…At the same time as the above developments demonstrate the relatively broad scope now available to design reporters to quantify additional physiological variables, other technical advances have made it easier to assemble DNA for new reporter constructs, to make transgenic organisms that contain the constructs, and quantify reporter output. Rapid assembly of reporter constructs is aided by improved in vitro DNA assembly methods and widespread adoption of construction by homologous recombination in yeast (Sands & Brent, ). Rapid construction of transgenic strains with well‐behaved reporters in C. elegans has been greatly aided by the development of methods that allow integration of reporters at defined chromosomal sites (Frokjaer‐Jensen et al ., ).…”

Methylase-assisted subcloning for high throughput BioBrick assembly

“…The costs of gene synthesis, cloning and DNA sequencing have decreased significantly but syntheses are still limited in length (≤3 kb), nucleotide composition, accuracy and yield ( Kosuri & Church, 2014 ; Kuhn et al, 2017 ). Many DNA assembly methods have been invented ( Casini et al, 2015 ; Chao, Yuan & Zhao, 2015 ; Sands & Brent, 2016 ; Vazquez-Vilar, Orzaez & Patron, 2018 ; Watson & Garcia-Nafria, 2019 ), which suggests that none work well for every user. The challenges of assembling cloned parts are not identical to those of ligating PCR products into plasmids ( Bryksin & Matsumura, 2010 ) so different solutions are demanded.…”