ABI have released MethylPrimer Express! - A new primer design program for DNA methylation....For Free!!! (Jun/02/2006 )

hi rosylight,

your sequence works fine here.

I pasted the sequence only and nothing else, the FASTA header will cause problems. There are no N's or X's in your sequence so that's fine.

I have posted the screenshots and it has picked some BSP primer sets for the 1.5kb CpG island found at the 5' end of this gene.

Good luck!

Nick

Hi,
Thanks for your help,karyotyper and Nick!

I'll restart my experiment with the new primers,and inform you of the results.


Best Wishes!

Hi,methylnick

I've used the primes designed by MPE to amplify the gene AF315385,but the results are frustrated.I think I should perform a nested PCR, so I have some questions to consult you .

The CpG island at 5' end of AF315385 is my target sequence ,ranging from 1 to 1426,so I should amplify it in the first round, then treat the PCR products with bisulfite, finally do the second round using treated DNA as template,right?

The external primers should be designed according to the original sequence,and can I use the MPE primers as internal primers?
Looking forward for your help

Best wishes

rosylight

rosylight,

a nested pcr strategy is favoured for bisulfite pcr and sequencing.

I noticed you are treating your pcr amplicon with bisulfite? This is incorrect as the DNA methylation marks are wiped after PCR amplification as Taq polymerase does not distinguish between a methylated and unmethylated cytosine. Typically, genomic DNA is treated with bisulfite and then PCR is performed.

I hope this clears things up a little.
Nick

Hola
Those words are in Spanish, it says : error number 6 in execution timing, overloading
good luck

Hi,methylnick

Thanks for your quick reply ,but I want more details.(In fact,I'm a medical doctor,so I'm not adept at molecular biology )

The primers designed by MPS are attached below. The aimed sequence is around the transcription start (5102),about 143bp. But the CpG island at 5' end is from 1 to 1426. Then what area should I choose to amplify in the first round PCR? And you tell me that genomic DNA is treated with bisulfite before PCR, that's to say , the sample DNA is single strand, are two sets of primers is needed in the first round? Which software is suitable for designing nested primes?


By the way ,could you tell me the PCR reaction you uesd to perform successfully?

Best wishes

Rosylight

Muchos Gracias chiocco!

Rosylight,

a nested PCR strategy is essentially picking two primer sets that flank your region of interest and one of the sets is internal to the other. This is essential for bisulfite PCR and sequencing (referred to here as BSP).

For MSP it usually is not necessary to do this. I am not too sure if you are interested in looking at the CpG island or the targeted region of interest selected here in methylprimer express because it sounds like the gene sequence is switched around from your description .

Take note that MSP only interrogates the methylation status of CpG's found within the primer sequences selected and this it maybe okay to infer methylation of the surrounding CpG's it needs to be checked!

Nick

Hi ,methylnick

I know that nested PCR needs two primer sets ,but I'm not sure wether the thing is the same after bisulfite treatment.And you've pointed out that nested PCR is not essential for MSP,so I think I don't have to consider it right now.

According to the ABI MPS guide and your explaination,I think I should choose the CpG islands around transcription start as my target.But I got no bands yet yesterday ,so I wonder what's going wrong,the primers or the conditions?Depending on your experience,do you think the primers designed by MPE are reasonable?And is AF315385 worthy of analyzing its methylation status?


Best Wishes
rosylight

rosylight,

could you tell me if you are trying to PCR using these primers on normal genomic DNA or genomic DNA that has been converted with bisulfite. The primers picked my methylprimer express for MSP are directed towards bisulfite converted DNA.

if you are using bisulfite converted DNA, then increasing the number of cycles in your PCR may work, or you may require an initial amplification step before using your MSP specific primers, if this is the case, I would then try to get MPE to design BSP primers (these are generic bisulfite primers not directed to methylated or unmethylated DNA) that encompass your MSP primer sets. So the initial amplification you would use the BSP primer set to generate enough material for a second PCR amplification using your MSP primer set that would enable you to see products on a gel.

Hope this helps

Nick

Nick

I use the genomic DNA converted with bisulfite.And I 've gotten BSP primers from MPE(the report is attched below),whose product is 439bp,including the target sequence of MSP primers(143bp).Hope I would get some product this time.

Thank you for your help very much !You are always so kind.

Best regards

rosylight