Degenerate Primers - (Mar/14/2007 )
I had a quick question. I'm planning on running sequencing analysis of bisulfite treated DNA and had a question about the design of the primers. I've seen different papers use different primer design parameters and I was curious as to what your opinions might be. Some of these papers designed their BSP primers so as not to include ANY possible methylation sites making them incapable of discriminating between methylated and unmethylated templates, whereas others seem to utilize degenerate primers (such as Y to recognize both C -methylated- or T -unmethylated-). Would the use of degenerate primers introduce a source of bias into the equation?
My greatest fear would be that the degenerate primer would bind preferentially to either methylated or unmethylated templates giving an inaccurate overview of the % methylation of the analyzed region.
So in your opinions, which of these scenarios would result in the most accurate data? or should they all essentially result in the same data?
1) Nested PCR - Degenerate bases used in both first round, 2nd round and sequencing primers.
2) Nested PCR - 1st round PCR using primers which do not include degenerate bases, 2nd round and sequencing primer include degenerate bases.
3) Nested PCR - 1st & 2nd round PCRs without degenerate bases in the primer. Sequencing primer contains degenerate bases.
4) Nested PCR - No degenerate bases used.
Is there a limitation on the number of degenerate bases which can be included in a primer?
A big problem with degenerate bases is the fact that they reduce the Tm of the primer. If you have lots of degeneracies, or if there are a few clustered near the 3' end of the primer, you will really destabilise the primer:template duplex, which will introduce the bias you mentioned. This is one good reason to try to avoid degeneracies in your PCR and sequencing primers.
You might be able to get by with running separate sets of reactions with non-degenerate primers in a Q-PCR reaction (which should give you ideas about the % methylation present). If your primers include a common primer sequence at their 5' end, you can use a single primer set for the sequencing stage.
I see absolutely no reason to believe that degenerate primers decrease melting temperature. They are simply mixtures of two or potentially many different distinct primers. These may have slightly different Tm's, but will not be significantly lower than the Tm of any other similar length primer sequences.
Sorry, but I have to disagree. Depending on the local sequence, a mismatch can drop the Tm by several degrees. If it's at the 3' end, this could show itself as a decreased efficiency of amplification in the first few critical cycles, which will introduce a bias. Once the amplicon has started to become significant, of course, such a mismatch effect will be removed as the sequence will be 100% identical, but by then, the 'damage' would have been done to the system.
Well, if there is a mismatch, of course the Tm will be lower. But the point of making degenerate primer mixtures is that you also have the matching primer in the mix. The matched primers will have the higher Tm, and will certainly preferentially be the ones which bind to the template strand.
OK, I was assuming the point of the expts was to look at relative levels of methylated and non-methylated DNA. In that case, wouldn't it be important to know that both types of DNA were being amplified at equal efficiencies. If that's not the case, then you are absolutely correct.
If using degenerate primers isn't an issue, then is there any reason why some researchers would go out of their way to ensure that their BSP primers contained no CpGs?
I have tried using degenerate primers before and the results seemed somewhat inconsistent with previous result using non-degenerate primers.
I amplified a 318bp region using non-degenerate primers and it was shown that >70% CpG sites were methylated in two cell lines, but then I amplified a 267bp region further downstream, which overlaps with the 318bp for 10 CpG sites, using degenerate primers, the results show that <20% CpG sites were methylated, and the previously methylated CpG sites are not methylated this time. I wonder if there is something to do with the degeneracies.
I remember there is a paper describing using touchdown PCR to overcome bias against unmethylated templates but it seems not working in my case. Has anyone encountered this before?
Thank you very much.
with degenerate primers you are assuming that there are equal proportions of your primer containing one base over the other, this is a very big assumption to make. I don't think this would be the case and you would inadvertantly introduce bias into the PCR if there are any deviations of your primer sequences.
This is why I stay away from designing degenerate primers.
You could start with two primers (say there is one CpG in the region where you want the primer) one with an A, the other a G. OD the primers and combine at an equal proportion. This would negate worrying about equal proportions. I would avoid using degenerate primers but if its not possible due to regions of low complexity on your DNA then degenerate primers are a must. I've seen them used in Nature papers before, although that doesn't prove there wasn't any bias...