For many researchers, PCR is routine. They don't give it a second thought. This DNA amplification technique has quickly become an essential tool in molecular biology. It is considered accurate, fast, and inexpensive.

"In my experience, PCR is basically problem-free. The machines exist. We use them, and they have proven to be reliable," says Eric F. Schmidt, a research associate at the Rockefeller University's Heintz Laboratory who works with bacterial artificial chromosome (BAC) transgenes.

Now, two researchers from Vanderbilt University have described a previously unknown barrier to PCR amplification, a DNA sequence of 370 nucleotides that forms a tight cluster of hairpin structures. This region is resistant to polymerase read-through, and, as a result, could lead to biased sequence data. All things considered, similar hairpin cluster sequences in the genome may also be resistant to PCR amplification, suggesting implications for sequencing and BAC recombineering.

The Foxd3 Hunt

At Vanderbilt University, associate professor Patricia Labosky studies the genetic controls governing embryonic development in mammals. Specifically, she’s interested in the Fox family of transcription factors, which play a role in regulating genes associated with cell growth, proliferation, and differentiation. Mutations in one gene in this family, Foxd3, are known to cause vitiligo, a condition that is characterized by depigmentation of the skin.

Last year, Labosky and Brian Nelms*, who was a postdoctoral fellow in her lab at the time, wanted to modify the Foxd3 locus to understand how certain mutations affected its function. So, they turned to BAC recombineering to generate DNA constructs.

Recombineering allowed them to quickly and efficiently construct vectors for subsequent manipulation and to manipulate the bacterial genome directly. In addition, the technique enhances functional genomics studies by providing better mouse models and a more refined genetic analysis of the mouse genome.

Today, BACs have become an important part of the molecular biologist’s toolbox. Because of their high stability and large insert size (100–300 kb), BACS have been used to sequence the genomes of several organisms. Now, BACs are being used to create transgenic mice since almost all of the important regulatory sequences that are required for normal gene expression can be placed on a single BAC. Also, laboratories are using BACs to make gene-targeting constructs to manipulate mouse genes using embryonic stem cell technology.

Hairpin in My Side

But there was a problem. During their attempts to produce the gene targeting vectors, Labosky and Nelms encountered a conserved stretch of 370 nucleotides in the 5' untranslated region of the Foxd3 locus that was resistant to polymerase read-through. In this region, one of the strands of DNA was predicted to extend out and formed a tight cluster of hairpins in the genome. This structure affected PCR amplification, sequencing, and recombination.

"We hit the same barrier no matter which approach we utilized," says Nelms. “But, this gave us an important clue, and ultimately we were able to come up with a solution to this barrier”

As Labosky and Nelms attempted to surmount the hairpin cluster, they eventually tried a primer designed from a sequence within the hairpin cluster. This primer allowed the polymerase to amplify a section of the hairpin cluster structure where the external primers could not penetrate. In the end, Nelms and Labosky amplified the entire hairpin cluster sequence in two pieces, using two primers from sequences within the hairpin cluster.

In a paper published in Scientific Reports in 2011 (1), Labosky and Nelms reported their struggles and eventual success with the hairpin cluster barrier in Foxd3 as a “cautionary note for researchers experiencing technical challenges with BAC recombineering or other molecular biology methods requiring recombination or other molecular biology methods requiring recombination or polymerase activity."

"I believe it is an interesting study in how scientists solve problems, both expected and unexpected, that come up during research studies. I am proud we found a solution to a problem that had not been previously addressed and hope that our paper will be of help to others doing research similar to ours," says Labosky.

Good to Know

In the end, Schmidt believes Labosky and Nelm’s paper will be useful for researchers. "If you are doing work which is dependent on just one DNA sample and run into a problem and cannot proceed, this is very good information to know," says Schmidt.

At the moment, Labosky is now continuing her investigation of the Fox family of transcription factors, having solved just one major barrier to understanding how these genes regulate development. Now, whether the 370-nuclotide hairpin cluster sequence of the Foxd3 gene actually serves a function in regulating the gene and controlling its expression remains to be determined.

References

1. Nelms, B. L., and P. A. Labosky. 2011. A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering. Scientific Reports 1.

* Nelms is currently assistant professor in the Biology Department at Fisk University and an adjunct assistant professor at the Vanderbilt University of Medicine.