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Curr Protoc Mol Biol. 2009 Apr;Chapter 3:Unit 3.21. doi: 10.1002/0471142727.mb0321s86.

DNA cloning and engineering by uracil excision.

Author information

1
New England Biolabs, Ipswich, Massachusetts, USA.

Abstract

This unit describes a simple and efficient DNA engineering method that combines nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning. PCR primers contain a single deoxyuracil residue (dU), and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions. The primers are then used to amplify DNA in discrete fragments that incorporate a dU at each end. Excision of deoxyuracils results in PCR fragments flanked by unique, overlapping, single-stranded extensions that allow the seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment. Two basic protocols on the methods of uracil excision-based engineering are presented, and special attention is given to primer design. The use of a commercially available cloning vector and the preparation of custom vectors are also presented.

PMID:
19343708
DOI:
10.1002/0471142727.mb0321s86
[Indexed for MEDLINE]

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