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A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering
Formation of higher-order structure of nucleic acids (hairpins or loops, for example) may impact not only gene regulation, but also molecular biology techniques and approaches critical for design and production of vectors needed for genetic engineering approaches. In the course of designing vectors...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255507/ https://www.ncbi.nlm.nih.gov/pubmed/22355623 http://dx.doi.org/10.1038/srep00106 |
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author | Nelms, Brian L. Labosky, Patricia A. |
author_facet | Nelms, Brian L. Labosky, Patricia A. |
author_sort | Nelms, Brian L. |
collection | PubMed |
description | Formation of higher-order structure of nucleic acids (hairpins or loops, for example) may impact not only gene regulation, but also molecular biology techniques and approaches critical for design and production of vectors needed for genetic engineering approaches. In the course of designing vectors aimed to modify the murine Foxd3 locus through homologous recombination in embryonic stem cells, we discovered a 370 nucleotide segment of DNA resistant to polymerase read-through. In addition to sequencing and PCR disruptions, we were unable to use BAC recombineering strategies to exchange sequences within the Foxd3 locus. This segment corresponds to a putative DNA hairpin region just upstream of the 5' untranslated region of Foxd3. This region is also highly conserved across vertebrate species, suggesting possible functional significance. Our findings provide a cautionary note for researchers experiencing technical challenges with BAC recombineering or other molecular biology methods requiring recombination or polymerase activity. |
format | Online Article Text |
id | pubmed-3255507 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-32555072012-01-11 A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering Nelms, Brian L. Labosky, Patricia A. Sci Rep Article Formation of higher-order structure of nucleic acids (hairpins or loops, for example) may impact not only gene regulation, but also molecular biology techniques and approaches critical for design and production of vectors needed for genetic engineering approaches. In the course of designing vectors aimed to modify the murine Foxd3 locus through homologous recombination in embryonic stem cells, we discovered a 370 nucleotide segment of DNA resistant to polymerase read-through. In addition to sequencing and PCR disruptions, we were unable to use BAC recombineering strategies to exchange sequences within the Foxd3 locus. This segment corresponds to a putative DNA hairpin region just upstream of the 5' untranslated region of Foxd3. This region is also highly conserved across vertebrate species, suggesting possible functional significance. Our findings provide a cautionary note for researchers experiencing technical challenges with BAC recombineering or other molecular biology methods requiring recombination or polymerase activity. Nature Publishing Group 2011-10-04 /pmc/articles/PMC3255507/ /pubmed/22355623 http://dx.doi.org/10.1038/srep00106 Text en Copyright © 2011, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by-nc-sa/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ |
spellingShingle | Article Nelms, Brian L. Labosky, Patricia A. A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering |
title | A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering |
title_full | A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering |
title_fullStr | A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering |
title_full_unstemmed | A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering |
title_short | A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering |
title_sort | predicted hairpin cluster correlates with barriers to pcr, sequencing and possibly bac recombineering |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255507/ https://www.ncbi.nlm.nih.gov/pubmed/22355623 http://dx.doi.org/10.1038/srep00106 |
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