A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering.

Scientific Reports
Brian L Nelms, Patricia A Labosky

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.

References

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Sep 1, 1997·Nucleic Acids Research·S F AltschulD J Lipman
Oct 17, 2002·Genes & Development·Lynn A HannaPatricia A Labosky
Mar 11, 2003·Nature Biotechnology·Giuseppe TestaA Francis Stewart
Feb 26, 2005·Nucleic Acids Research·Søren WarmingNeal G Copeland
Mar 28, 2008·Development·Lu TengPatricia A Labosky
Apr 22, 2008·Nucleic Acids Research·Andreas R GruberIvo L Hofacker
Aug 21, 2008·Methods in Molecular Biology·Nicholas R Markham, Michael Zuker
Feb 6, 2010·Cytopathology : Official Journal of the British Society for Clinical Cytology·E D RossiG Fadda
Jul 31, 2010·The FEBS Journal·Julian L Huppert

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Datasets Mentioned

BETA
GM12878

Methods Mentioned

BETA
PCR
biopsies
restriction digests
ChIP-Seq
electrophoresis

Software Mentioned

UNAFold
ECR browser
RNAfold
BLAST
DCODE ECR browser
Basic Local Alignment Search Tool ( BLAST )
Ensembl genome browser
DCODE
DCODE ECR Browser Genome Browser
ECR

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