There’s a lot of talk out there about bisulfite conversion, how useful it is, how it’s the “Gold Standard,” how laborious it is, how rough it is on your samples, but the reality of it is that there probably isn’t any better way to prep your samples for single nucleotide resolution methylation profiling, so don’t be intimidated by your lab-mate who had a bad experience with conversion a couple years ago. Isn’t that guy all thumbs anyway?
We caught up with Zymo Research, a team that has truly taken the “bs” out of bisulfite, leaving you with “iulfite” (don’t ask us how to pronounce it) and more time to focus on getting results. These scientists have performed more bisulfite conversion preps than most researchers will in a lifetime. Follow a few guidelines at these critical steps and you’ll be talking more about your interesting data than complaining about a lab technique, which is cool because nobody likes a complainer.
Denature of the Beast
Bisulfite conversion only works efficiently on single stranded DNA, so the DNA denaturation step is key. Both chemical and heat based methods can be effectively used to denature the DNA for bisulfite conversion. Earlier methods often use sodium hydroxide to beat DNA into single-stranded submission prior to the conversion process, but more recent protocols turn up the heat by bringing the entire conversion reaction to 98C for a bit before moving to a longer incubation at cooler temps. Heat denaturation gives you the benefit of a faster, more streamlined procedure while taking place in the presence of the bisulfite; so the conversion process begins immediately once the strands start separating.
Avoid a Bad (Conversion) Reaction
The bisulfite conversion reaction is where the magic happens, changing cytosine to uracil. The great thing about bisulfite is that these sequence changes can be easily read by a number of downstream technologies. The downside is there’s really no “spell checker” when it comes to downstream analysis, so you’ll want to ensure that your reactions are doing their job. According to Seth Ruga, an R&D Staff Scientist at Zymo, here are some things to look out for:
- Low Bisulfite Concentration
- Too Much DNA In Conversion Reaction
“Low bisulfite and high DNA concentrations cause incomplete conversion for similar reasons; by interfering with the ratio of bisulfite to DNA,” added Ruga. When these concentrations are not optimized, the reaction can start to go hay-wire, so double check those dilutions and make sure to follow your protocol to keep everything dialed in.High bisulfite concentrations at low pH causing degradation.? But…? That’s right; the exact conditions where conversion works best can cause significant damage to the DNA. Like a bad break-up, sample degradation will leave you in pieces (or with pieces anyway)… really small ones that are difficult to analyze.
“Today’s protocols and kits minimize degradation in the conversion process mainly through buffer formulation and designing incubation parameters to yield the highest conversion efficiencies with the least amount of damage to the DNA,” explained Ruga. It is also important, both for degradation and recovery, that the starting material for the conversion reaction be as high quality as possible, degraded starting DNA will only make the problem worse during the conversion.” As they say, “garbage in. garbage out”.
- Under-Conversion and Over-Conversion
“Incomplete conversion of non-methylated cytosine and the over conversion of methylated cytosine are two potential consequences of the bisulfite conversion process that will mostly effect data analysis,” shared Larry Jia, R&D Director at Zymo. Complete conversion will change your sequence from CTCT to TTTT. But if it’s under-converted it may look like TTCT. These generally occur when there has been incomplete denaturation or the DNA-to-bislufite reaction ratio is off. An over-conversion event may make your CT-MeC-T sequence into TTTT when it should be TTCT. It can be tricky to pin down over-conversions since they happen pretty rarely, and are impossible to distinguish from a methylation error by the methyltransferase, resulting in a chemical/enzyme finger pointing session that isn’t too productive.
If you follow the guidelines for the denaturing and conversion steps, you should have plenty of your sample to recover. Historically, poor recovery of bisulfite converted DNA was largely due to poor optimization and consistency of “home brew” conversion reactions combined with laborious, time consuming purification methods. Nowadays, sample clean-up is pretty streamlined so assuming you weren’t too soft, or didn’t go overboard on one of the earlier steps you should be in good shape for cleaning up your sample.
That said, keep in mind that the DNA post-conversion will be cytosine depleted and largely single stranded since the strands are no longer complementary. This means that the DNA is much more sensitive and should be stored with appropriate caution, so don’t expect your DNA to have a shelf-life like SPAM® afterwards.
Ruga offered up a quick QC of your sample. “The degree of degradation in any given reaction can be easily checked by running a converted DNA sample on an agarose gel and then chilling the gel to visualize the DNA. Generally, there will be a smear from about 100-200bp up to 1-2kb.”
Conversion kits out there now have addressed these issues with tricks like coupling conversions with column based purification and desulphonation techniques. It’s now easy to get recovery of over 80% of the converted DNA from the conversion and elute it in very small volumes to keep concentrations high.
Go Forth and Convert Away!
Bisulfite conversion is one of the epigenetics researcher’s biggest balancing acts. Too little, too much, too long, too short will all have you troubleshooting and generally looking up at the sky and asking “why?!” Your lab may want to tough it out with that old protocol from the 80’s, but we encourage you to check out an ultra-optimized commercial option. They take the guesswork out of the process and give you access to some solid tech support who are paid to care about your technical issues, unlike your lab or roommates who will all be making a move for the door at the first sign of trouble.
Big thanks to Seth Ruga and Larry Jia and our other friends at Zymo Research for passing along some of their bisulfite expertise.
Zymo’s Bisulfite Products have seen more citations than an episode of Traffic Cops so if you’re looking to bulletproof your upcoming bisulfite preps, check out their many bisulfite conversion kits at their site.