Bisulfite conversion can be used in so many different applications that it’s not so surprisingly referred to as the gold standard for DNA methylation analysis. But getting to the gold can create some problems for DNA. The process of converting methylated DNA (5mC) to Uracil and then finally a Thymine is harsh as it involves the application of heat and basic conditions.
These conditions lead to DNA damage and the loss of precious samples. On the flip-side, using milder conditions leads to incomplete conversion and the presence of the intermediate dhU6S. Both sides of the coin bias PCR as Taq polymerase struggles to get past the bulky modified bases of damaged DNA or the conversion intermediates.
While Taq polymerase has long been the champion of PCR, the lab of Phillip Holliger previously developed another polymerase (5D4) by directed evolution to tackle hydrophobic base analogues to enable an expanded genetic code. Now they set out to characterize whether their polymerase would perform as well on the bulky bases that can arise from varying bisulfite conversion conditions.
Here’s what went down:
- The team created a small template containing cytosines in various combinations.
- They then tested a library of polymerase mutants, including some created by directed evolution, on DNA from different bisulfite conversion conditions.
- Most polymerases performed poorly, although 5D4 took the trophy on damaged DNA and DNA containing the conversion intermediate dhU6S.
- Changing templates, 5D4 chugged right past stretches of U without exhibiting the stalling typical of Taq and thus substantially outperformed Taq in regions of high GC content.
- 5D4 appears to be better at not only ‘reading’ converted DNA but also better at utilizing non-canonical bases as a template during polymerization.
5D4 can instantly be swapped into any existing workflows and has a lot of potential in the study of rare samples, such as certain stem cell populations and precious tissue bank samples.
The team concludes with their optimistic outlook that further directed evolution using bisulfite converted DNA as a substrate could lead to even greater efficiency and sensitivity of 5D4 on bisulfite converted DNA, as these bases were not the original target.
Learn more about getting this evolved polymerase into your workflow over at Nucleic Acids Research, August 2015