Novel method for the scarless single‐step assembly of many small DNA sequences

Abstract

Techniques in the assembly of DNA fragments have expanded rapidly, enabling complex and efficient assemblies that traditional restriction digest based assembly cannot accomplish. However, the scarless assembly of multiple small (<1kb) DNA fragments remains difficult and time consuming, especially a problem as most genetically modified proteins are constructed on this scale. Here we present a novel method using a cycled ligation reaction to assembly many small DNA fragments into a transformation ready plasmid. First, the reaction is heated to a denaturing temperature. Disposable Oligonuleotide Connectors (DOCs), oligonulceotides ~40 bp in length that are homologous to the end of one insert sequence and the beginning of the next then act as a guide, bringing the end and beginning of two insert strands together when the reaction is decreased to an annealing temperature. This allows for a ligase to ligate the two insert strands together, in a sequence exactly determined by the user with no scar sequences. The reaction is then cycled between denaturing and annealing/ligating temperatures, allowing the ligated products of early cycle to serve as a template for further ligations in later cycles. We have successfully assembled as many as 10 ~150 bp inserts into a transformable plasmid in a single reaction. DOC‐based Assembly represents a powerful new tool for the construction of complex genetic constructs.