Short homologies efficiently generate detectable homologous recombination events

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15bp. Although the use...

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Bibliographic Details
Published in:Analytical biochemistry 2014-10, Vol.462, p.26-28
Main Authors: Osahor, Andrew N., Tan, Chau-Yan, Sim, Edmund Ui-Hang, Lee, Choon-Weng, Narayanan, Kumaran
Format: Article
Language:English
Subjects:
BACs
Base Sequence
Chromosome
Chromosomes, Artificial, Bacterial - genetics
E. coli
Escherichia coli - genetics
Genetic Engineering - methods
Homologous Recombination
Homology
Linear
Plasmids - genetics
Recombineering
Sequence Homology, Nucleic Acid
Telomere - genetics
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Summary:When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2014.05.030