The bisulfite conversion experts from Zymo Research have put together a killer guide on the ins and outs of amplifying bisulfite treated DNA. EpiGenie got an exclusive, advanced peek at some of the content that we are sharing with our readers. In this section, they provide some helpful advice on the art of bisulfite primer design:
Optimizing Bisulfite Primer Design
Optimizing primers for bisulfite amplification can be challenging at first, but if you follow a few guidelines, you should be on your way to successful results relatively quickly. There are two types of primer design approaches depending on your application; Bisulfite PCR primers, which will amplify a region regardless of methylation status, and Methylation Specific PCR (MSP) primers which are designed to amplify only when a target locus is either methylated or unmethylated:
Primer Design for Bisulfite PCR – In this scenario, primers are used, as they are in other PCR settings, to amplify the template, most often for analysis by another downstream methodology (e.g. bisulfite sequencing, mass spectrophotometry) that will determine the DNA methylation status.
Unlike normal PCR where your template contains 4 bases, bisulfite PCR primers need to be longer to contend with the loss of most of the cytosines. Shoot for primers of at least 26-30 bases of sufficient specificity. Also, since your template will be highly fragmented, we recommend targeting an amplicon size between 150-300 bp.
Next, you will want to make sure that the primers avoid CpG sites, or if that is not possible; try to locate them at the 5’ end of the primer with a mixed base at the cytosine position.
When designing the primers, it is a good exercise to ‘convert’ the DNA first – changing all of the non-CpG cytosines to uracils. It is also important to note that only one strand of the template will be amplified by a given primer set. Because the DNA strands are no longer complementary, only the reverse primer will actually bind to the target DNA. The forward primer will only have a complementary template once generated from the reverse primer. Usually, 35 to 40 cycles are required for successful amplification.
Primer Design for Methylation Specific PCR (MSP) – Unlike primers for bisulfite PCR, MSP primers serve the dual functions of amplifying bisulfite converted DNA and interrogating the DNA methylation status at specific CpG sites.
In addition to observing the above practices for standard bisulfite amplification, MSP primers should be designed taking the following guidelines into account:
- CpG site(s) of interest must be included in the primers.
- The CpG(s) should be located at 3’ end of the primers.
- Two primer sets are required for each amplicon that will be interrogated
- A “Methylated” primer set that contains cytosine (C) at CpG sites
- And, a “Non-methylated” primer set that uses thymine (T) in place of C at CpG locations.
(A) Following bisulfite treatment, the two converted strands of the DNA template are no longer complementary.
(B) Primers for Bisulfite PCR are designed for subsequent sequencing and analysis of cytosines within the amplicon. CpG sites within the primers should be avoided or located at the 5′-end with a mixed base at the cytosine position (Y= C/T, R= G/A). Sequencing data is commonly represented by a “lollipop” plot where closed circles represent methylated cytosine positions and open circles non-methylated ones.
(C) Primers for Methylation Specific PCR (MSP) are designed to target and assess the methylation status at specific CpG sites. CpG sites within the primers must be located at the 3’-end to increase their specificity to methylated (M) or non-methylated (U) templates. Completely methylated or non-methylated templates will generate a single amplicon from only their representative primer set following MSP. Samples with mixed methythation, will be amplified by both primer sets.
Use a Hot Start Polymerase
When amplifying bisulfite-converted DNA, it is ideal to use a hot start polymerase. Non-specific amplification is relatively common with bisulfite-treated DNA due to its AT-rich nature.
A hot start polymerase will minimize primer dimerization and the non-specific amplification products that go along with them, better than regular Taq polymerase.
Turn Up the Heat with Higher Annealing Temperatures
At Zymo Research, we have found that higher annealing temperatures are key for good amplification, and work outside our lab, including that of M.D. Anderson’s Lanlan Shen, (Biotechniques, 2007) also confirmed that raising the annealing temperatures was instrumental in improving PCR efficiency from bisulfite converted templates.
The good news is that longer primers suggested for bisulfite primer design, not only help with specificity, but also bring your melting temperature up. It’s also best to include as many guanines as possible in the primer regions to increase the melting temperature of the primer to the recommended 55-60°C range.
When first trying out a new primer set, we suggest running an annealing temperature gradient. This will help you identify the ideal annealing temperature to use in your experiments. Troubleshooting your primers before starting your PCR analysis will save you time and eliminate any non-specific results.
** Coming Soon!: A Comprehensive Guide to Bisulfite-converted DNA Amplification will be available for download in the EpiGenie Guide section or from the Zymo Research website**