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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Bibliographic Details
Published in:Scientific reports 2011-10, Vol.1 (1), p.106-106, Article 106
Main Authors: Nelms, Brian L., Labosky, Patricia A.
Format: Article
Language:English
Subjects:
5' Untranslated Regions
631/114
631/1647
631/337
631/532
Animals
Bacterial artificial chromosomes
Base Sequence
Chromosomes, Artificial, Bacterial
Cluster Analysis
Deoxyribonucleic acid
DNA
DNA Primers
DNA sequencing
Embryo cells
Forkhead Transcription Factors - genetics
Gene regulation
Genetic engineering
Homologous recombination
Humanities and Social Sciences
Humans
Molecular biology
multidisciplinary
Nucleic acids
Polymerase chain reaction
Polymerase Chain Reaction - methods
Recombination, Genetic
Science
Science (multidisciplinary)
Sequence Analysis, DNA - methods
Stem cell transplantation
Stem cells
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep00106