Recombineering Linear BACs

Part of the Methods in Molecular Biology book series (MIMB, volume 1227)


Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

Key words

Linear BAC Recombineering E. coli Genomic DNA Chromosome Phage N15 Plasmid 



The authors are grateful to Nikolai Ravin for providing N15 reagents and to Sek-Chuen Chow for support and encouragement. Q.C. is grateful to Monash University Malaysia for a HDR Scholarship. This work was partly funded by a Fundamental Research Grant Scheme FRGS/1/2011/ST/MUSM/02/2 from the Ministry of Higher Education Malaysia to K.N.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.School of ScienceMonash University MalaysiaBandar SunwayMalaysia
  2. 2.Monash University MalaysiaBandar SunwayMalaysia
  3. 3.Icahn School of Medicine at Mount SinaiNew YorkUSA

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