Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/111630
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Type: Journal article
Title: Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
Author: Ratnayake, D.
Newman, M.
Lardelli, M.
Citation: BMC Research Notes, 2018; 11(1):202-1-202-7
Publisher: BioMed Central
Issue Date: 2018
ISSN: 1756-0500
1756-0500
Statement of
Responsibility: 
Dhanushika Ratnayake, Morgan Newman and Michael Lardelli
Abstract: Objective: Repeat expansion of polyglutamine tracks leads to a group of inherited human neurodegenerative disorders. Studying such repetitive sequences is required to gain insight into the pathophysiology of these diseases. PCR-based manipulation of repetitive sequences, however, is challenging due to the absence of unique primer binding sites or the generation of non-specific products. Results: We have utilised the degeneracy of the genetic code to generate a polyglutamine sequence with low repeat similarity. This strategy allowed us to use conventional PCR to generate multiple constructs with approximately defined numbers of glutamine repeats. We then used these constructs to measure the in vivo variation in autophagic degradation activity related to the different numbers of glutamine repeats, providing an example of their applicability to study repeat expansion diseases. Our simple and easily generalised method of generating low repetition DNA sequences coding for uniform stretches of amino acid residues provides a strategy for generating particular lengths of polyglutamine tracts using standard PCR and cloning protocols.
Keywords: Polyglutamine repeats; codon redundancy; degeneracy of the genetic code; PCR based repeat region amplification; aggregate proteins; autophagy
Rights: © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
RMID: 0030084955
DOI: 10.1186/s13104-018-3298-5
Grant ID: http://purl.org/au-research/grants/nhmrc/1061006
Appears in Collections:Molecular and Biomedical Science publications

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