At EpiGenie, we try to call attention not just to the big names in the field that nab most of the limelight, but also to the folks busting their pipettes day and night for new discoveries. So why not give a shout out to a workhorse in the most widely used method for methylation analysis, bisulfite sequencing. Polymerases are rarely the stars of presentations nowadays. Instead, it seems that the “box” (the sequencer) gets all of the attention. We understand–it’s the closest thing to a Ferrari most labs will ever see–but what about the gas (polymerases) in the tank?
To amplify bisulfite-converted sequences, researchers need an accurate polymerase that can handle deoxyuracil. Most proofreading polymerases stall out after they incorporate the base. On the other hand, Taq polymerase, the old PCR standby, efficiently incorporates deoxyuracil but has a nasty error rate, introducing a mutation once every 125,000 nucleotides or so. Until recently, researchers had to put up with Taq’s sloppiness—which can be significant at the genome-wide scale—if they wanted to do bisulfite sequencing.
The Polymerase that Won’t Get Stuck on U
That all changed when researchers recently picked up PfuTurbo Cx Hotstart DNA Polymerase, a proofreading polymerase that not only knows what to do with deoxyuracil, but also has high proofreading activity. A mutant form of Pfu polymerase, PfuTurbo Cx Hotstart DNA polymerase plows right past deoxyuracil with an error rate of only about 1 in 780,000 bases, making it ideal for downstream apps. like bisulfite sequencing.
With all the innovation in the sequencing arena, it’s about time that Taq was traded in for a newer, flashier model. Researchers are starting to embrace PfuTurbo Cx (the name even sounds like a sports car), as seen in a recent Methods paper on Reduced Representation Bisulfite Sequencing (RRBS) of mammalian genomes.
Turbo-Charged Reduced Representation Bisulfite Sequencing
In this approach, introduced by MIT’s Alexander Meissner, researchers digest genomic DNA with methylation-insensitive restriction enzymes that recognize CpG-containing motifs. Then, they ligate sequencing adaptors to the fragments and size-select CpG-dense regions on a gel for bisulfite conversion, amplification, and sequencing.
A critical step in the RRBS procedure is PCR amplification of the bisulfite-converted sequences, and according to the authors, “the choice of polymerase is essential.” The team notes that “Pfu Turbo Cx Hotstart (Stratagene) is an attractive option as it provides high-rate read-through of uracyl bases but retains additional proofreading activity to reduce mutation.”
So the next time you get all starry-eyed over the latest and greatest sequencer, take a minute to think outside “the box” about the polymerases busting their active sites to get you your data.