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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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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. |
format | Online Article Text |
id | pubmed-5870680 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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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