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Methods Enzymol. 2013;533:157-77. doi: 10.1016/B978-0-12-420067-8.00010-6.

Recombineering: highly efficient in vivo genetic engineering using single-strand oligos.

Author information

1
Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA. Electronic address: sawitzkj@mail.nih.gov.

Abstract

Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10kb) deletions, and small (10-20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations.

KEYWORDS:

Bacterial outgrowth; Designing the oligo for recombineering; Double-strand DNA (dsDNA); In vivo genetic engineering; Methyl-directed mismatch repair (MMR) system; Single-strand DNA (ssDNA); Single-strand oligos

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