Restriction Enzyme-Mediated DNA Family Shuffling

Behrendorff, James B. Y. H, Johnston, Wayne A. and Gillam, Elizabeth M. J (2014) Restriction Enzyme-Mediated DNA Family Shuffling. Methods in Molecular Biology, 1179 175-187. doi:10.1007/978-1-4939-1053-3_12

Author Behrendorff, James B. Y. H
Johnston, Wayne A.
Gillam, Elizabeth M. J
Title Restriction Enzyme-Mediated DNA Family Shuffling
Journal name Methods in Molecular Biology   Check publisher's open access policy
ISSN 1064-3745
ISBN 9781493910526
Publication date 2014
Year available 2014
Sub-type Article (original research)
DOI 10.1007/978-1-4939-1053-3_12
Open Access Status
Volume 1179
Start page 175
End page 187
Total pages 13
Place of publication New York, NY United States
Publisher Humana Press, Inc.
Collection year 2015
Language eng
Formatted abstract
DNA shuffling is an established recombinatorial method that was originally developed to increase the speed of directed evolution experiments beyond what could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length variants of the original gene that have inherited mutations from multiple templates. Subsequent studies demonstrated that directed evolution could be further accelerated by shuffling similar native protein-coding sequences from the same gene family, rather than mutated variants of a single gene. Generally at least 65–75 % global identity between parental sequences is required in DNA family shuffling, with recombination mostly occurring at sites with at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I digestion and avoids the risk that parental sequences may be over-digested into unusable very small fragments. Restriction enzyme-mediated fragmentation also reduces the occurrence of undigested parental sequences that would otherwise reduce the number of unique variants in the resulting library. In the current chapter, we provide a brief overview of the alternative methods currently available for DNA shuffling as well as a protocol presented here that improves on several previous implementations of restriction enzyme-mediated DNA family shuffling, in particular with regard to purification of DNA fragments for reassembly.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

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Created: Fri, 21 Nov 2014, 10:27:33 EST by Mrs Louise Nimwegen on behalf of Aust Institute for Bioengineering & Nanotechnology