Here we describe a DNA sequence of 370 nt resistant to polymerase read-through, resulting in precise DNA sequencing stops and preventing PCR amplification across this region. This region is conserved and predicted to form secondary structure comprised of a cluster of hairpins. Regions of sequence that produce sequencing stops are usually attributed to GC-rich regions or secondary structure, but consideration of such structures in the design of vectors has been generally under-studied and further analyses would be necessary to systematically determine if there is a defined effect of secondary structure on homologous recombination at other loci or with other techniques. The presence of this type of region or secondary structure may be important to consider in other genes, especially if it has the potential to interfere with attempts to amplify these sequences by PCR or execute recombination across these regions, such as during BAC recombineering. We also acknowledge the possibility that the predicted secondary structure could interfere with any of the other components needed for homologous recombination in addition to DNA polymerase13
. In previously constructed Foxd3
targeting vectors this area was cloned using restriction enzyme sites located outside the 370 nt sequence, avoiding the need for PCR amplification or homologous recombination in E. coli14,15
. Although BAC recombineering and PCR at this specific area of the Foxd3
locus was unsuccessful, and the mechanism for hindrance of BAC recombineering is not precisely defined, we show here that another potential strategy to overcome the inability to PCR-amplify specific segments of polymerase-resistant DNA is to start from the middle and generate two amplicons that can later be pieced together. This may be an important alternative for researchers encountering difficulties with these commonly used polymerase-dependent approaches.
Our observation and analysis of conservation of this region is consistent with the possibility that the predicted secondary structure may also have functional significance. This region may be important as part of the Foxd3
promoter or an enhancer element that regulates Foxd3
transcription in specific cell types. Interestingly, aligning the same sequence analyzed within the DCODE ECR Browser with tracks from the ENCODE (Encyclopedia of DNA Elements) Project using the UCSC Genome Browser showed the conserved 370 nt region
corresponded to peaks in trimethylation of Histone 3 Lysine 4 (H3K4me3) and DNase hypersensitivity, both indicators of open chromatin in two mesoderm-derived cell lines (GM12878 and K562) (). This correlation could also explain the sequence conservation we observed. It is unknown whether this predicted structure occurs in vivo
, where recruitment of specific DNA or histone binding factors could stabilize or eliminate higher-order structures formed in this area. Although further functional analysis of this 370 nt segment is necessary to determine whether it impacts Foxd3
gene expression, we have identified an intriguing conserved element and offer the interesting idea that this potential DNA secondary structure may have functional significance.