Application of the Restriction-Free (RF) cloning for multicomponents assembly

Methods Mol Biol. 2014;1116:73-87. doi: 10.1007/978-1-62703-764-8_6.


Molecular manipulations, including DNA cloning and mutagenesis, are currently employed on a routine basis in all life science disciplines. Over the last decade new methodologies have emerged that expanded and facilitated the applications for DNA cloning. The classical Ligation-Dependent Cloning (LDC) is gradually replaced by Ligation-Independent Cloning (LIC) techniques. The Restriction-Free (RF) cloning was originally developed for introduction of a foreign DNA into a plasmid at any desired position. The RF methodology is based on generation of a PCR product, which serves as a set of mega-primers for subsequent incorporation into any desired position within a circular plasmid. We have expanded the applications of the RF methodology for multiple simultaneous alterations of a target DNA and for multicomponents assembly. In the current manuscript we describe a step-by-step protocol for application of the RF methodology for simultaneous multiple DNA fragments assembly in tandem and at distinct positions within an expression vector.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular / methods*
  • DNA, Circular / genetics
  • Escherichia coli / genetics
  • Genetic Vectors / genetics


  • DNA, Circular