Genetic Engineering by DNA Recombineering
Name
nihms-1033634.pdf
Description
Accepted version
Size
1.53 MB
Format
Adobe PDF
Checksum (MD5)
24d53111599859dc3593c97d06352501
Author(s)
Papa, Louis John
Shoulders, Matthew D.
Date Issued
September 2019
Journal
Current Protocols in Chemical Biology
Publisher
Wiley
Citation
Papa, Louis John and Matthew D. Shoulders. "Genetic Engineering by DNA Recombineering." Current Protocols in Chemical Biology 11, 3 (September 2019): e70 doi 10.1002/CPCH.70 ©2019 Author(s)
Version
Author's final manuscript
Abstract
Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the cassette to its intended location. These homology arms can be easily introduced as 5' primer overhangs during the PCR reaction. The flexibility to choose almost any pair of homology arms enables the precise modification of virtually any DNA for purposes of sequence deletion, replacement, insertion, or point mutation. Recombineering often offers significant advantages relative to previous homologous recombination methods that require the construction of cassettes with large homology arms, and relative to traditional cloning methods that become intractable for large plasmids or DNA sequences. However, the tremendous number of variables, options, and pitfalls that can be encountered when designing and performing a recombineering protocol for the first time introduce barriers that can make recombineering a challenging technique for new users to adopt. This article focuses on three recombineering protocols we have found to be particularly robust, providing a detailed guide for choosing the simplest recombineering method for a given application and for performing and troubleshooting experiments.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1002/CPCH.70
