Some of the biggest issues associated with bisulfite conversion aren’t really related to the bisulfite conversion process itself, but rather the downstream assays interpret the base conversion. After all, if a C is converted to a U in a test tube and nothing can assay it effectively, was in really converted? OK, enough bisulfite philosophy.
The complexity reduction that accompanies the bisulfite conversion process introduces a few challenges to sample preparation methods like PCR, so let’s take a look at how you can make the most of it when amplifying your sample.
Hot Start Things Off with the Right Mix
Ever since Kary Mullis unleashed PCR into labs in the early 80’s, DNA polymerases like Taq have been put to work in labs all over the world. Over the years new polymerases have been developed and commercialized to address some of the tougher amplifying scenarios like bisulfite treated DNA.
Hot start polymerases are a good example. These heat activated beauties keep primer dimers at bay more effectively than regular Taq while limiting non-specific amplification products that can cause headaches. “Using a hot-start polymerase helps by preventing the polymerase from being active during the PCR setup period, when primers at room temperature are more prone to nonspecific binding,” according to Lam Nguyen, R&D Scientist at Zymo Research.
Not all mixes have been optimized for templates like bisulfite converted samples. Make sure the hot start mix you’re thinking about using has been used to amplify bisulfite converted DNA already or you might have to put in a little time with optimization. We asked the Zymo Research R&D staff to share what’s involved with creating a PCR mix that’s optimized for amplifying bisulfite converted templates.
“We optimized the buffer conditions after conducting many titrations of known PCR boosters and other salt constituents that lower annealing temperature, reduce primer dimerization, and limit non-specific binding from occurring when working with bisulfite treated samples,” explained Nguyen.
“Then we screened every available hot start polymerase we could find that delivered the most specific and the most robust amplification with bisulfite converted DNA.”
So, if you like optimization work then consider that approach. If not, then check out some of the hot starts out there and make sure your amplification reaction is up to snuff.
Bisulfite Primer Design Simplified
Of course, the other key ingredient in PCR is primers. Nobody really pays attention to primers until there’s a lot on the line and things aren’t working well. Bisulfite conversion doesn’t help the thankless role of primers. The complexity reduction makes designing high specificity primers challenging. Zymo R&D scientist Ron Leavitt offered up a stepwise path for designing primers:
“When designing the primers from the known DNA sequence, it is a good exercise to ‘convert’ the DNA first – changing all of the non-CpG cytosines to uracils. The first primer should be designed to base-pair with this converted sequence (normally this would be thought of as the reverse primer). The second (forward) primer should be designed to base-pair to the extension of the first primer and not the “opposite strand” like in traditional PCR. Primers for bisulfite PCR normally range from 23-35 bases, with amplicon sizes generally ranging from 200-500 bases.”
Or, if you want to get automated, check out these Primer Design resources:
- MethPrimer: Pop in your sequence of interest and out come some recommended primer sequences. Nice.
- methPrimerDB: Scope this database out for validated primers for various DNA methylation analysis method
Turn Up the Heat
Higher annealing temperatures are key for good amplification. Leavitt added, “The longer than normal primers are necessary to aid in specificity to the template and to bring melting temperature up. Bisulfite primers should have Tms greater than or equal to 50oC. It is best to include as many guanines as possible in the primer regions to increase the melting temperature of the primer. Additional bases can also be added to the primer to bring up the melting temperature. For unbiased amplification, each primer can contain at most one CpG dinucleotide. If the primer does contain a CpG, use the mixed bases purine (A or G, usually abbreviated ‘R’ and used in the first primer) and pyrimidine (C or T, usually abbreviated ‘Y’ and used in the second primer) so that both methylated and non-methylated DNAs are amplified.”
Leavitt’s not alone when it comes to the importance of high annealing temps. Some recent work lead by M.D. Anderson’s Lanlan Shan (Biotechniques, 2007) found that raising the annealing temps was instrumental in improving PCR efficiency in bisulfite templates.
“Additionally, ending the primer with a converted C (from the original sequence) has been shown to increase the specificity of the reaction. Lastly, when using a new set of primers, consider running an annealing temperature gradient spanning the calculated temperature to determine where the primers are performing most efficiently,” added Leavitt.
Primed for Bisulfite Success
Regardless of your downstream application, chances are you’ll need to amplify your converted DNA at some point. If you find yourself staring at a long list of variables to troubleshoot and are becoming more superstitious (well it did during the last full moon…) about how to get good results, check out these simple rules first.
Huge thanks to Lam Nguyen and Ron Leavitt, R&D Scientists over at Zymo Research for the solid tips. You probably know about Zymo’s Bisulfite Kits, but if you’re looking for a solid hot start mix, check out ZymoTaq which was designed for bisulfite converted templates.