title: A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering.
abstract: 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.
authors.affiliation: Vanderbilt University Medical Center, Department of Cell and Developmental Biology, Center for Stem Cell Biology, Program in Developmental Biology, Nashville, Tennessee, USA.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Nelms
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Labosky
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D016000:Cluster Analysis; D017931:DNA Primers; D051858:Forkhead Transcription Factors / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D020121:5' Untranslated Regions; D017931:DNA Primers; C503514:FOXD3 protein, human; D051858:Forkhead Transcription Factors