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Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats

We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using ϕ29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat...

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Detalles Bibliográficos
Autores principales: Osborne, Robert J., Thornton, Charles A.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275075/
https://www.ncbi.nlm.nih.gov/pubmed/18263610
http://dx.doi.org/10.1093/nar/gkn025
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author Osborne, Robert J.
Thornton, Charles A.
author_facet Osborne, Robert J.
Thornton, Charles A.
author_sort Osborne, Robert J.
collection PubMed
description We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using ϕ29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and ϕ29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG(exp)) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.
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spelling pubmed-22750752008-04-07 Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats Osborne, Robert J. Thornton, Charles A. Nucleic Acids Res Methods Online We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using ϕ29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and ϕ29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG(exp)) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat. Oxford University Press 2008-03 2008-02-07 /pmc/articles/PMC2275075/ /pubmed/18263610 http://dx.doi.org/10.1093/nar/gkn025 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Osborne, Robert J.
Thornton, Charles A.
Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
title Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
title_full Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
title_fullStr Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
title_full_unstemmed Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
title_short Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
title_sort cell-free cloning of highly expanded ctg repeats by amplification of dimerized expanded repeats
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275075/
https://www.ncbi.nlm.nih.gov/pubmed/18263610
http://dx.doi.org/10.1093/nar/gkn025
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