Intact recombineering of highly repetitive DNA requires reduced induction of recombination enzymes and improved host viability.
Recombineering technology permits flexible engineering of large DNA in Escherichia coli without dependence on suitably placed restriction sites. However, recombineering is limited for modifying highly repetitive DNA because of its potential to trigger instability by uncontrolled self-recombination of the repeats. In this study, induction of the recombineering enzymes and growth condition of the host are optimized to demonstrate intact modification of bacterial artificial chromosomes (BACs) containing long arrays of centromeric alpha satellite repeats. This optimized recombineering protocol may be useful for manipulation of other biologically important repetitive DNAs, including trinucleotide repeat expansions and homologous gene families, to facilitate their functional studies.
Narayanan
Kumaran
K
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA. kumaran.narayanan@mssm.edu
D002470:Cell Survival; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004790:Enzyme Induction / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
D011994:Recombinant Proteins; D004247:DNA