designing primer for methylation - (Apr/23/2006 )
sunshine1981,
it is possible to do MSP on a gene that has not been done before, however it would be prudent to perform BSP on the gene you are interested in and then design an MSP primer set that allows you to quickly type your samples as methylated or unmethylated.
Nick
Jim006,
one way of doing this is to make dupilcate MSP reactions and take one out (stop the PCR) at 10 cycles, another at 15 cycles, 20 cycles and then 25 cycles and then run them on a gel. This would cover all bases.
Nick
one way of doing this is to make dupilcate MSP reactions and take one out (stop the PCR) at 10 cycles, another at 15 cycles, 20 cycles and then 25 cycles and then run them on a gel. This would cover all bases.
Nick
Thanks for your quick answer,nick.
DO you mean that I fix my first round cycle number(35),and adjust the cycle number of the second round.
Jim006
Jim,
no just for the second round, I am not too sure your primer design setup. Is the initial round just a non-specific (in terms of methylation) to obtain enough amplicon for the second round (methylation specific?)
if so, then it's the second round that you do this.,
Nick
Nick:thankyou verymuch!
could you tell me how to purchase the agent :NaHSO3,hydroquinol,i know them be better bought from sigma.can you tell me more details. i find i did not still know buy what !help me !thank you !
Sodium bisulfite (5)
CAS Number: 7631-90-5
243973 ACS reagent, mixture of NaHSO3 and Na2S2O5 (Sigma-Aldrich)
S9000 ReagentPlus™, ≥99% (Sigma-Aldrich)
28-1980 SAJ first grade, ≥58.5% as SnO2 (SAJ)
28-1970 JIS special grade, 64.0-67.4% as SO2 (SAJ)
S8890 ACS reagent (Sigma)
Hydroquinone (10)
Linear Formula: C6H4-1,4-(OH)2, Formula Weight: 110.11, CAS Number: 123-31-9
H9003 ReagentPlus™, ≥99% (Sigma)
H3660 meets USP testing specifications (Sigma-Aldrich)
H7148 suitable for photographic applications, ≥99% (Sigma-Aldrich)
53960 puriss., ≥99.0% (HPLC) (Fluka)
15616 puriss., ≥99.5% (HPLC) (Riedel-de Haën)
53965 purum, ≥98.0% (HPLC) (Fluka)
H17902 ReagentPlus™, 99% (Sigma-Aldrich)
13-1930 SAJ special grade, ≥99.0% (SAJ)
13-1940 SAJ first grade, ≥99.0% (SAJ)
240125 ≥99% (Aldrich)
Nick :
Another question:if the other agents are not very important and purchasing them wherever is ok?
tell me all you know 
thankyou very much!
and you suggest me should do BSP first,then i should conside if the DNA is treated by NaHSO3 completely,i have no control ,how could i do ?i am looking forward your repliation,thankyou ! 
no just for the second round, I am not too sure your primer design setup. Is the initial round just a non-specific (in terms of methylation) to obtain enough amplicon for the second round (methylation specific?)
if so, then it's the second round that you do this.,
Nick
Thanks nick.
As the protocol,two rounds of pcr is ideal and I could not get any bands after the first round of 35 cycles.And we know that nested-MSP will enhance the specificity of pcr greatly.
Jim
Hi,Purplefetus.Thank you for your reply.
Well,FHIT gene is a tumor suppressor gene.As what you have said above,tumor suppressor genes are normally unmethylated,in healthy people.I donot know why I often amplified the methy-bands in healthy people' PBMC DNA.I am sure that there is no contamination when extracting the DNA.How do you design your primers?By methprimer?Have you runinto the same promblem?
jim006
Hey Jim,
Sorry for the late reply. I have not done MSP. I've only attempted BSP and was successful so far. I like BSP because I think a more definitive answer is reached. You get to see the actual sequence and then base your ideas on that. I'm afraid of doing MSP since I hear that sometimes bands come out with both sets of primers, sometimes with none. I'm also evaluating a large # of CpG's therefore it would take me forever to analyze my promotor region using MSP. I would need to design atleast a dozen primers. I used a combination of perl primer and methprimer to design my primers. I also eyeballed some of them out myself. So it was a combination of all three.
Hello,
Just wanted to add my two cents. Our lab originally started methylation analysis trying to work out methods for melting curve analysis.
In theory melting curve analysis should easily be able discriminate methylated from unmethylated DNA. If you design primers to only amplify a few CpG sites (very difficult) using bisulfite sequencing primers (by which I mean no CpG sites recognized by the primer). You can then perform melting analysis and determine how many methylated sites you have with the lightcyler. Higher CG (methylated) would equate to higher melting temperatures. Many papers claim that you can discriminate a single methylated site by a temperature change of .25 degrees. By creating a standard curve of known methylation (induced by enzyme methylation) you can determine how many sites you have methylated in your sample.
One of the major issues we ran into is that the melting curve analysis is very sequence specific. Designing primers can be a major pain in the ass. The more sites you have the less information you can glean from the melting analysis. Also if the product you are trying to amplify has a lot of AT runs or TTTTTTTT sequences, which is common after bisulfite treatment. What ends up occuring is that the product will give a very odd melting pattern resulting in multiple peaks which are nearly impossible to analyze. We have since switched gears to methyl-light, and pyrosequencing and haven't looked back.
-CW
Hi,Purplefetus.Thank you for your reply.
Well,FHIT gene is a tumor suppressor gene.As what you have said above,tumor suppressor genes are normally unmethylated,in healthy people.I donot know why I often amplified the methy-bands in healthy people' PBMC DNA.I am sure that there is no contamination when extracting the DNA.How do you design your primers?By methprimer?Have you runinto the same promblem?
jim006
Hey Jim,
Sorry for the late reply. I have not done MSP. I've only attempted BSP and was successful so far. I like BSP because I think a more definitive answer is reached. You get to see the actual sequence and then base your ideas on that. I'm afraid of doing MSP since I hear that sometimes bands come out with both sets of primers, sometimes with none. I'm also evaluating a large # of CpG's therefore it would take me forever to analyze my promotor region using MSP. I would need to design atleast a dozen primers. I used a combination of perl primer and methprimer to design my primers. I also eyeballed some of them out myself. So it was a combination of all three.
Thank you for your reply,purplefetus.
Yes,BSP is a golden criterion to discover the methylation status of the gDNA. But it wastes much time and money.I am studying the clinical samples,MSP is better for me,which is quick ,specific and sensitive.After all,Thanks a lot.
Jim