Pyrosequencing imprinted genes - (Am i thinking straight?) :( (Aug/10/2006 )
I have just got a Pyrosequencing assay design for an imprinted gene that I have looked at with MSP. (Didn't get consistent PCR results!)
My pyrosequencing assay encompasses the methylated PCR product of MSP.
Am I right in thinking the methylated primer will not work in the MSP if it cann't bind to the converted sequence (indicating LOI).
On pyrosequencing I will be able to determined the presence or absence of methylation at the CpG's I assay based on their sequence.
So despite not looking at the unmethylated region of my MSP - I should be able to determine if my CpG's around the MSP methylated primers are unmethylated. This should tell me if I have the maternal/paternal allele. Right?
I'm not sure I got that out right - basically since in MSP I look at regions that are 1300bp apart I was wondering if I need another Pyrosequencing assay around the unmethylated region of my MSP? Will that help confirm results better?
Makes sense to anyone???
Sorry learning, I don't quite understand what you're trying to do.
Given appropriate primer design your M and U primers will amplify methylated and unmethylated DNA respectively. Assuming your normal control shows both bands by MS PCR then the absense of either in your test samples indicates LOI (in humans usually by deletion, UPD or imprinting centre mutation and occasionally by other epigenetic means). Your MS PCR result should give you the answer.
Sequencing the result of an imprinted M PCR product should give you a M result. The primers determine the sequence amplified so I can't see the point of sequencing this PCR product.
Have I understood you correctly???
Ok I guess I was not clear.
I have run my MSP and yes my M & U primers will both amplify & give me a product incase of an imprinted gene, and a missing band would indicate LOI.
When looking at solid tumors normal cell contamination can contribute to the PCR product formation. So I decide to use pyrosequencing - based on the C's that get converted to T's and are not present in CpG dinucleotides their software can determine the % of C=T conversions or C=C retention at CpG's.
So now my Pyrosequencing primers have been designed such that it includes the region that I looked at with my M primer (not the uprimer) of my MSP.
I assume if there is methylation I will get close to 100% C=C retention and if unmethylated close to 100% C=T conversion.
Since my MSP primers were designed ~1300 bp apart I guess my concern was does looking at just the M region good enough?
Sorry learning I still don't get it.
I assume if you use M primers and sequence you will get results based on DNA amplified from these primers only - is that what you want? If there has been LOI then your M primers either won't work or there will be reduced product if some methylation sequence homology remains, but I still don't see how this helps you (although there may be some evidence of a mosaic pattern in amplified M DNA as opposed to a uniform M pattern if this normally occurs). Shouldn't you be trying to determine a shift in the total proportion of methylated to unmethylated DNA?
I'm also not sure if I've misinterpreted your comments on pyrosequencing - are you saying that CpG methylation is determined by examining the rate of conversion of non-CpG C's to T's???
You wrote: "So I decide to use pyrosequencing - based on the C's that get converted to T's and are not present in CpG dinucleotides their software can determine the % of C=T conversions or C=C retention at CpG's".
Sorry I can't help further. Maybe Methylnick or PCRMan can help - or you could try speaking to the pyrosequencing rep. I gather you're working on H19/IGF2 or something similar.
hmmm firstly learning,
I think your MSP primer design may not be rather efficient as the primers amplify a 1300bp region? Typical MSP assays amplify 100-300bp of sequence to return a result of methylated or unmethylated by the presence of a PCR amplicon using methylation specific primers. so for an inprinted region, you will find amplicons in both M and U primer sets, if one set fails to give a band then there is possibility of LOI.
My understanding of pyrosequencing is that it will sample every CpG site within a region of interest and will give you a proportion of methylation at a given CpG site, very much like direct sequencing in BSP. You can from the results determine LOI if you have deviations from 50% methylation at each CpG within your region.
As for determining the origin of the LOI, you would need to look for informative polymorphisms within your region that could identify for you whether the methylation signals were derived from the father or the mother and the methylation cannot be assigned to either using pyrosequencing, you would need to goto BSP for this as it will give you the full methylation pattern from each "allele".
Well, I guess for some reason I am unable to clearly get my point across. One more shot!
I have 2 different methods - MSP where M primer amplifies region A ~120bp and U primer amplifies region B ~180bp. A and B are 1300 KB apart.
Now my Pryrosequencing primers (different from MSP primers) includes/overlaps with the region A amplified in MSP by the M primer.
Pyrosequencing will be able to give me the % of C=T or C=C conversions at CpG's.
Methylated CpG's will show 100% C=C in the sequencing while unmethylated CpG's will show 100% C=T.
Like Nick mentioned imprinted genes will be expected to show 50% C=T & C=C at the CpG's.
My concern was that since my pyrosequencing includes only the region detected by M primer in MSP and the U primer region B is 1300bp downstream. Will I still be getting reliable imprinting information?
And to answer my question I guess I will! Since a DMR can be large and I will know if their is no methylation at the amplified A region...(MSP again) in my sequencing!!!
I hope I haven't confused people even more!!!
Sorry LEarning I am still a little confused. For a true MSP assay you need to design M and U primers to the same region. I am not sure why you have selected M and U primer sets as you have stated.
To obtain reliable imprinting information it would depend on your DMR, if it has been characterised previously then it would be okay to do it your way, however if it is not, then you would need to assay the entire 1300-1500bp region to fully guage imprinting status.
I still would like to stress that if your region is differentially methylated, you can not tell this simply by pyrosequencing, you would need to perform bisulfite sequencing and seqeunce a handful of clones where you would see clones either completely hypomethylated or completely hypermethyated. a 50% signal from pyrosequening could suggest variable methylation at all CpG's across the region that average to give a 50:50 result.
Hi again learning
Your last explanation was very clear and I understand what you're trying to do now.
I have to agree with methylnick that your MSP design is not ideal as you are assaying two completely different areas. For example, the region where your M primers is located could be differentially methylated so you will obtain an M PCR product. The region where your U primers is located might be hypomethylated and you will obtain a U PCR product. You cannot therefore assume that everything in between the M and U primers is differentially methylated (in this example one end is actually differentially methylated and the other is hypomethylated). This is not such an issue if the area you are studying has been well characterised by others (which sounds like it might be the case) but you should ideally have overlap between the two sets of primers if this is not the case.
Your pyrosequencing data will be relevant to the area you are sequencing (around the M primers) and as nick says, if this region is well characterised, you should be able to interpret the data accordingly (approx. 50:50 methylated to unmethylated expected). Some studies use densitometry to demonstrate deviation from a 1:1 methylated to unmethylated ratio. Generally a shift in the parental imprint can be proven by looking at informative polymorphisms following BSP or by looking for evidence of the presence of an informative exonic SNP following RT PCR and sequencing.
Hi Karyotyper and Nick,
Thanks for your inputs - yes this is a well characterised region and I will be pyrosequencing Bisulphite treated DNA (just incase i wasnt clear about that!).
This has also been a lesson in good scientifc writing I try