Primer design for MSP queries - (Dec/07/2004 )
Hi there,
I'm hoping to perform MSP to determine whether several genes of interest are methylated or unmethylated in my cells. I'm new to the field of DNA methylation and would very much appreciate the opportunity to ask a few (dumb!) questions:
1. MethPrimer appears to be a particularly user-friendly program by which to design primers for MSP. I have read that the optimal area for MSP primer selection is the most G-C rich region closest to the transcription start site. What sequence should I enter into MethPrimer to design my primers? I was thinking the 1000bp before the first exon of the gene, but wondered whether I should actually also include the first exon. Or perhaps two searches to produce two sets of primers: one on the 1000bp before the first exon, and one on the first exon??
2. In addition to the M and U primer sets, I have also read of the use of a third primer set denoted "W" (wild-type) that serves as a positive control for PCR, ie. it anneals to any DNA unmodified by bisulfite. The original papers outlining the technique described this method but most that have followed have only used M and U primers. How important are "W" primers? Because my bisulfite treatment for my cells will need to optimized, I was thinking it would be prudent and include some "W" primers. How are "W" primers designed? MethPrimer doesn't seem to have this option and only returns M and U primers.
3. I have noticed that Chemicon (who makes the CpGenome DNA Modification Kit I have) also provides CpG Ware software to design primers for MSP. How does this compare to MethPrimer? I entered the same sequence into both programs and got vastly different primers designed. MethPrimer appears to give a clearer, more informative output of the sequence entered, but CpG Ware appears to also give some options for "W" primers, as well as M and U.
4. I have also read of the need to use both positive and negative DNA samples previously determined to be methylated and unmethylated, respectively. I have no such samples first-hand and was curious as to what I could use. Is there something 'standard' (CpGenome Universal Methylated DNA?) or is it best to try and repeat some published findings? I have read of the intense study of the p16 and E-cadherin tumor suppressor genes and wondered whether I would need to go to the lengths of using two of the same cell lines which were found to be differently methylated, as well as the same primers, to optimize MSP in my hands before moving on to the unknowns? I would ideally like to work with just the promoter/first exon regions of my genes of interest and so would be encouraged for my own future studies to see that primers designed in these regions of p16 or E-cadherin with either MethPrimer or CpG Ware gave the same differences in methylation as those published primers which may not have used these programs to design primers.
Primer design appears to be the most critical parameter affecting the specifity of MSP, hence all my primer concerns above. I greatly appreciated all replies, hopefully these queries are relatively straightforward. And I apologize in advance if I've asked too many questions.
thank you,
jamie
Hi Jamie
Let me try to answer your questions.
>>1. MethPrimer appears to be a particularly user-friendly program by which to design primers for MSP. I have read that the optimal area for MSP primer selection is the most G-C rich region closest to the transcription start site. What sequence should I enter into MethPrimer to design my primers? I was thinking the 1000bp before the first exon of the gene, but wondered whether I should actually also include the first exon. Or perhaps two searches to produce two sets of primers: one on the 1000bp before the first exon, and one on the first exon??
Based on my research, I think that only methtylation within a CpG island region is biologically significant in terms of its effect on transcription. For example, I mapped the E-cadherin gene methylation in two cell lines with one expresses Ecad and the other doesn't. Clearly a region immediately upstream the CpG island is methylated in both cells while only the Ecad negative cell has methylation within the CpG island. Since more than 60% of genes have CpG island in their promoter region, and most CpG islands are at the proximal promoter region and some may extend into the first exon, the first step of primer design is to decide where the CpG island is. If there is no CpG island surrounding the transcriptional start site, or near it, I probably won't touch that gene. You can first paste a big sequence including the 5'-flanking region and first exon into the MethPrimer program to check if there is any CpG island, and then pick primers on the CpG island by selecting "Pick primer on CpG island" (if there is only one) or by specifying a target region which is within the CpG island.
>>2. In addition to the M and U primer sets, I have also read of the use of a third primer set denoted "W" (wild-type) that serves as a positive control for PCR, ie. it anneals to any DNA unmodified by bisulfite. The original papers outlining the technique described this method but most that have followed have only used M and U primers. How important are "W" primers? Because my bisulfite treatment for my cells will need to optimized, I was thinking it would be prudent and include some "W" primers. How are "W" primers designed? MethPrimer doesn't seem to have this option and only returns M and U primers.
"W" primers serve as controls for non-specific binding of primers and for completeness of DNA conversion. You don't necessarily need the W primers, instead, as a control, you can also use the M and U primer to amplify unmodified DNA. In my opinion, even if W priemrs give a negative result, it doesn't guarantee that DNA conversion is 100% complete, because the C residues (resistant to bisulfite) are quite unevenly distributed along DNA strands.
>>3. I have noticed that Chemicon (who makes the CpGenome DNA Modification Kit I have) also provides CpG Ware software to design primers for MSP. How does this compare to MethPrimer? I entered the same sequence into both programs and got vastly different primers designed. MethPrimer appears to give a clearer, more informative output of the sequence entered, but CpG Ware appears to also give some options for "W" primers, as well as M and U.
Never tried CpG ware.
>>4. I have also read of the need to use both positive and negative DNA samples previously determined to be methylated and unmethylated, respectively. I have no such samples first-hand and was curious as to what I could use. Is there something 'standard' (CpGenome Universal Methylated DNA?) or is it best to try and repeat some published findings? I have read of the intense study of the p16 and E-cadherin tumor suppressor genes and wondered whether I would need to go to the lengths of using two of the same cell lines which were found to be differently methylated, as well as the same primers, to optimize MSP in my hands before moving on to the unknowns? I would ideally like to work with just the promoter/first exon regions of my genes of interest and so would be encouraged for my own future studies to see that primers designed in these regions of p16 or E-cadherin with either MethPrimer or CpG Ware gave the same differences in methylation as those published primers which may not have used these programs to design primers.
For MSP, the bisulfite modification and subsquent PCR amplification are also challenging. To start, you can use some type of kits such as the one from Chemicon. Other things may also help: known working primers, known working and modified DNA, well-estabolished model system (a confirmed methylated gene is a cell line). If noboy has studied the gene you are interested, just go ahead and work on that gene.
>>Primer design appears to be the most critical parameter affecting the specifity of MSP
I agree. Also critical are PCR conditions. If they are not optimized or even "optimized" you will probably get "partial methylation" for every samples you are going to analyze (band for both M and U).
Good luck with your adventure.
Hi pcrman,
Thanks for your quick reply, it was very helpful. You effectively answered all my queries, but at the same time raised one other. This concerns when you said that "only methylation within a CpG island region is biologically significant in terms of its effect on transcription", and then cited your experience with methylation of an E-cadherin CpG island versus a region upstream, correlating this with the gene's expression in different cell lines.
Unfortunately, not all of my genes of interest have CpG islands in their promoter regions (1000 bp upstream of exon 1) and exon 1. This is according to the output from MethPrimer using default settings. However, largely all possess CpG 'sites' (ie. dinucleotides) and it is upon these that MethPrimer appears to have designed MSP primers when there is no island. Is my interpretation of MethPrimer here correct? More importantly, is studying methylation this way possible?... that is, if one wishes to determine whether methylation state correlates with gene expression. I note you mentioned that "if there is no CpG island surrounding the transcriptional start site, or near it, I probably won't touch the gene" (I presume you mean here by MSP). However, in my case I am unfortunately restricted in what genes I can study. From a few papers I've read in the literature, the methylation state of individual CpG sites (as determined not by MSP but by bisulfite sequencing) appears to correlate well with gene expression in cells where one would expect these genes to be expressed. However, it is logical that islands - where there is a concentration of sites - are "more reliable" markers for methylation state.
I guess what I'm also effectively asking here is that if around 60% of human genes have CpG islands in their promoter regions, how can you assess the promoter methylation state of the other 40% of genes? Most CpG papers I've seen mention the magical 60% value, largely because they are focusing on the islands, but don't dwell too much on the rest. Should bisulfite sequencing be used in this instance? Is MSP at all informative? To your knowledge has MSP been successfully applied to the analysis of methylation in genes without CpG islands but only CpG dinucleotides??
I thank you again in advance for all your help... and for the well wishes (I will need them!)
jamie
Hi Jamie,
You have raised a question which is quite confusing to all in the field. My understanding of current concept about DNA methylation is: In normal situation, the majority of cytosines are methylated except those within the CpG island which is associated with transcriptional regulatory region of most genes; while in cancer cells, some CpG islands become methylated resulting silencing of associated gene and the bulk DNA sequence may be hypomethylated. So if you want to study a gene which doesn't have a CpG island in its promoter region, even you can find some methylation to the sparsely distributed CpG SITEs, the methylation most likely has nothing to do with the expression status of the gene. That is why I said if I couldn't find a CpG island for a gene, I wouldn't bother to stduy its methylation because it doesn't have an effect on the gene's transcription. The problem is many people just randomly pick a gene and map some CpG sites for methylation in the promoter and then claim that the gene is methylated in cancer.
No matter what method you use, I doubt the meaning of stduying methylation of a gene without a CpG island.
I also think MSP can only be applied to CpG sites whose methylation status has been defined to be important to transcription using other high resolution methods such as bisulfite seqeuncing.
I hope that helps.