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Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences

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...

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Detalles Bibliográficos
Autores principales: Ratnayake, Dhanushika, Newman, Morgan, Lardelli, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870680/
https://www.ncbi.nlm.nih.gov/pubmed/29587822
http://dx.doi.org/10.1186/s13104-018-3298-5
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author Ratnayake, Dhanushika
Newman, Morgan
Lardelli, Michael
author_facet Ratnayake, Dhanushika
Newman, Morgan
Lardelli, Michael
author_sort Ratnayake, Dhanushika
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13104-018-3298-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-58706802018-03-29 Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences Ratnayake, Dhanushika Newman, Morgan Lardelli, Michael BMC Res Notes Research Note 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13104-018-3298-5) contains supplementary material, which is available to authorized users. BioMed Central 2018-03-27 /pmc/articles/PMC5870680/ /pubmed/29587822 http://dx.doi.org/10.1186/s13104-018-3298-5 Text en © The Author(s) 2018 Open AccessThis 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.
spellingShingle Research Note
Ratnayake, Dhanushika
Newman, Morgan
Lardelli, Michael
Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
title Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
title_full Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
title_fullStr Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
title_full_unstemmed Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
title_short Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
title_sort degenerate codon mixing for pcr-based manipulation of highly repetitive sequences
topic Research Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870680/
https://www.ncbi.nlm.nih.gov/pubmed/29587822
http://dx.doi.org/10.1186/s13104-018-3298-5
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