A rapid and simple method for DNA engineering using cycled ligation assembly.

DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex se...

Full description

Bibliographic Details
Main Authors: Theodore L Roth, Ljiljana Milenkovic, Matthew P Scott
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2014-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC4167330?pdf=render
id doaj-8900726c24c244c1b591dcf5fbb4a58d
record_format Article
spelling doaj-8900726c24c244c1b591dcf5fbb4a58d2020-11-25T02:34:21ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0199e10732910.1371/journal.pone.0107329A rapid and simple method for DNA engineering using cycled ligation assembly.Theodore L RothLjiljana MilenkovicMatthew P ScottDNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method's utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.http://europepmc.org/articles/PMC4167330?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Theodore L Roth
Ljiljana Milenkovic
Matthew P Scott
spellingShingle Theodore L Roth
Ljiljana Milenkovic
Matthew P Scott
A rapid and simple method for DNA engineering using cycled ligation assembly.
PLoS ONE
author_facet Theodore L Roth
Ljiljana Milenkovic
Matthew P Scott
author_sort Theodore L Roth
title A rapid and simple method for DNA engineering using cycled ligation assembly.
title_short A rapid and simple method for DNA engineering using cycled ligation assembly.
title_full A rapid and simple method for DNA engineering using cycled ligation assembly.
title_fullStr A rapid and simple method for DNA engineering using cycled ligation assembly.
title_full_unstemmed A rapid and simple method for DNA engineering using cycled ligation assembly.
title_sort rapid and simple method for dna engineering using cycled ligation assembly.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2014-01-01
description DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method's utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.
url http://europepmc.org/articles/PMC4167330?pdf=render
work_keys_str_mv AT theodorelroth arapidandsimplemethodfordnaengineeringusingcycledligationassembly
AT ljiljanamilenkovic arapidandsimplemethodfordnaengineeringusingcycledligationassembly
AT matthewpscott arapidandsimplemethodfordnaengineeringusingcycledligationassembly
AT theodorelroth rapidandsimplemethodfordnaengineeringusingcycledligationassembly
AT ljiljanamilenkovic rapidandsimplemethodfordnaengineeringusingcycledligationassembly
AT matthewpscott rapidandsimplemethodfordnaengineeringusingcycledligationassembly
_version_ 1724809428084981760