Application of the Restriction-Free (RF) Cloning for Multicomponents Assembly
- 10 December 2013
- book chapter
- Published by Springer Science and Business Media LLC
- Vol. 1116, 73-87
- https://doi.org/10.1007/978-1-62703-764-8_6
Abstract
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.Keywords
This publication has 15 references indexed in Scilit:
- SLIC: A Method for Sequence- and Ligation-Independent CloningPublished by Springer Science and Business Media LLC ,2012
- Transfer-PCR (TPCR): A highway for DNA cloning and protein engineeringJournal of Structural Biology, 2011
- Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expressionJournal of Structural Biology, 2010
- Enzymatic assembly of DNA molecules up to several hundred kilobasesNature Methods, 2009
- High Throughput Production of Recombinant Human Proteins for CrystallographyMethods in molecular biology (Clifton, N.J.), 2008
- A versatile ligation-independent cloning method suitable for high-throughput expression screening applicationsNucleic Acids Research, 2007
- Harnessing homologous recombination in vitro to generate recombinant DNA via SLICNature Methods, 2007
- RF cloning: A restriction-free method for inserting target genes into plasmidsJournal of Biochemical and Biophysical Methods, 2006
- An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmidsProtein Expression and Purification, 2005
- Ligation-independent cloning of PCR products (LIC-PCR)Nucleic Acids Research, 1990