A Rapid and Simple Method for DNA Engineering using Cycled Ligation Assembly

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Abstract/Contents

Abstract: 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 I 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. In tests I 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. I applied cycled ligation assembly to construct chimeric proteins, revealing functional roles for individual domains of the Hedgehog signaling pathway protein PTCH1. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.

Type of resource	text
Date created	June 15, 2014

Author	Roth, Theodore L.
Primary advisor	Scott, Matthew
Advisor	Nelson, James
Degree granting institution	Stanford University, Department of Biology, 2014

Location	https://purl.stanford.edu/mk055zj2651

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License: This work is licensed under a Creative Commons Attribution Share Alike 3.0 Unported license (CC BY-SA).

Preferred Citation: Roth, Theodore L. (2014). A Rapid and Simple Method for DNA Engineering using Cycled Ligation Assembly. Stanford Digital Repository. Available at: http://purl.stanford.edu/mk055zj2651

Undergraduate Theses, Department of Biology, 2013-2014

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