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Differences between MSP and BSP-DNA Methylation, Histon

关键词: 2008-07-08 00:00 来源:互联网 点击次数:4536

Hello everyone I'm new here and I was wondering if I could get some assistance.

In my research of the literature I thought the differences between the two methods were based on the proximity of the primers to the CpG sites and that BSP primers were designed without including CpG dinucleotides.

I was wondering if someone could clear it up for me and what are the pros and cons of each method. Thank you very much in advance

-gungrave-

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Hi Gungrave,

I assume by BSP you mean bisulfite PCR and sequencing. If so, the advantages of this over MSP are:

A greater number of CpG residues analysed for methylation when compared with MSP (MSP is only one and that is identified with a methylation specific primer). Bisulfite sequencing looks at every CpG residue across the whole amplicon.

I would also say that primer optimisation is crucial for an MSP assay to work, it is not so crucial for BSP because you will be sequencing through the amplicon anyway.

MSP has it advantages in that it is far more quicker than BSP in terms of hands-on lab time.

hope this helps. I would be interested to see what other people think about this also.

Cheers

nick

-methylnick-

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Thanks for the reply. I always thought that with MSP one could include a number of Cpg dinucleotides in the region being amplified especially in the 3' end primer design to make the primer more specific.

Does this mean we only look at one CpG dinucleotide with MSP and if we want to observe several then use BSP?

My confusion started today when I ran across a paper that described the MSP they conducted by using restriction enzyme isoschizomers and amplifying the region that the enzymes cut. Thanks once again.

-gungrave-

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QUOTE(gungrave @ Feb 28 2005, 11:29 PM)

Does this mean we only look at one CpG dinucleotide with MSP and if we want to observe several then use BSP?

  My confusion started today when I ran across a paper that described the MSP they conducted by using restriction enzyme isoschizomers and amplifying the region that the enzymes cut. Thanks once again.

I suppose it is possible to look at multiple CpGs with MSP primers. Just clone and sequence the amplicons.

MSP has advantages in that you are able to gauge the methylation status by detecting the amplicon. An MSP primer usually ends to discriminate either a methylated or unmethylated C so it is a really quick way to assaying for methylation.

I think you are referring to COBRA which stands for combined bisulfite and restriction analysis. This is like the midway between MSP and BSP, COBRA is limited to the recognition site of the restriction enzyme, so you can potentially assy for more than one CG residue, but you are not assaying all of them.

Cheers

Nick

-methylnick-

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I agree with Nick-- MSP, even combined with a BstI restriction of the amplicon, is called site specific; it only lets you look at however many CpGs are in your primers and in a BstI site if you are lucky enough to have one. I wanted to use this method as well to avoid sequencing and cloning but in the end I have determined that sequencing is the only thing I trust and I tend to scoff at papers who use MSP as it is so prone to artifact. Check out my agarose bead protocol in the "bisulfite treatment" thread, pinned above.

Good luck,

Ellen

-labtechie-

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Thank you all for for your replies, there's one more thing and I hope I'm not beating a dead horse, bear with me if so .

The differences I see in MSP and BSP come from the primer design where MSP primers are designed with CpG(better in the 3' end) to make them more specific.

BSP on the other hand, primers are designed to encompass the CpG without including them in the design of the primers.

Does that sound about right? Thank you all very much

-gungrave-

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That is right-- for BSP, you want NO CpGs in your primers, and convert all non-CpG Cs into Ts in the forward primers (you figure out the reverse). BSP should amplify all converted strands regardless of methylation status, then it's the sequencing that matters. But do you see how you get a much better picture of the whole island this way?

-labtechie-

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gotcha, thanks for clearing that up

-gungrave-

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I second Ellen about MSP. If PCR conditions and primer design are not optimized, its result is prone to artifact. I simply won't believe any PCR method that relies on one nucleotide to avoid mispairing, even the nucleotide is at the 3' end of the primer.

-pcrman-

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I second Ellen about MSP. If PCR conditions and primer design are not optimized, its result is prone to artifact.  I simply won't believe any PCR method that relies on one nucleotide to avoid mispairing, even the nucleotide is at the 3' end of the primer.

I very agree about that...

-Ming-

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BSP on the other hand, primers are designed to encompass the CpG without including them in the design of the primers.

Hi everyone,

Cheers and good luck!!

nick

-methylnick-

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There is a bisulfite primer design tool called bisearch which can design such degenerate primers at bisearch.enzim.hu

-pcrman-

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Hi,methlnick,Thanks for your help.

Sorry I am the newcomer to this Forum ,I could not find where to put my attachment in "my control panel".So I send the attachment to the commen address of the Bioforum, wish it would be transfer to you smoothly.

Of course ,the primers for bisulfit sequencing are different from M and U primers in MSP;In fact in the PCR for Bisulfite sequencing,I follow your protocol and also lower the annealing T ,change the Mg++ concentration,but there is nothing, 3.0/4.3/6.7 mM Mg concentratiin (which works with M and U primer,product <200bp) did not work in this PCR (product should be 505bp)only gived the primer dimer.Even my French boss can not figure out a way.Would you please have a look at the attachment and give me the suggestion?

-biofemme-

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Biofemme,

here is your file with a new primer seqeunce added to it.

Basically for bisulfite PCR you require two rounds of PCR to obtain the product, you are normally amplifying a larger region than MSP so the larger templates would be more scarce as it is known bisulfite degrades the DNA during the conversion process.

Good luck with it, all the best!

Nick

-methylnick-

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Is BSP easier than MSP since after bisulfite treatment you can just PCR and sequence the PCR prouct? Did anyone use Methprimer for BSP primers design?

-hn37041-

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for BSP you are able to assay a greater number of CG sites. It is of the same difficulty as MSP and primer design is a crucial factor!

I have used methprimer before but I would like to caution you when using them, I got artefactual results and sequences that just didn't make sense. I might suggest you try perlprimer I think it does a better job that methprimer in BSP primers.

good luck!

-methylnick-

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Thanks for your help!

I will take a look at the perlprimer.

QUOTE(methylnick @ Jun 7 2005, 03:51 PM)

for BSP you are able to assay a greater number of CG sites. It is of the same difficulty as MSP and primer design is a crucial factor!

I have used methprimer before but I would like to caution you when using them, I got artefactual results and sequences that just didn't make sense. I might suggest you try perlprimer I think it does a better job that methprimer in BSP primers.

good luck!

[snapback]16827[/snapback]

-hn37041-

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One more question. I guess I have to use the restriction enzyme to digest DNA first. What is the rule to pick the enzyme except not cut the region you are going to PCR?

Thanks!

-hn37041-

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QUOTE

I guess I have to use the restriction enzyme to digest DNA first. What is the rule to pick the enzyme except not cut the region you are going to PCR?

It is not a must but an option. You can cut with any enzymes that don't cut within the amplified region.

-pcrman-

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another way that is simpler is to shear your DNA by running it through a 21g syringe needle about ten times. This will reduce the complexity of gDNA you are using and also help when strands need to be denatured fully for bisulfite treatment.

Nick

-methylnick-

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Thanks all for your help!

-hn37041-

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If I do enzyme digestion before bisulfite, do I need to purify the DNA after digestion?

-hn37041-

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I guess better clean up the DNA before modification. It's said pure DNA is important for complete conversion. check one of the pinned threads you will find the method how to do the clean-up.

-pcrman-

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That depends,

BSP is it's pro's and con's as does MSP. BSP can assay every CpG within your amplicon, MSP cannot.

MSP requires only two PCRs, one for methhylated and one for unmethylated and you get your result.

BSP only requires PCR and either direct sequencing or cloning and sequencing. These steps are more than just PCR. Direct sequencing is a little challenging and if it works it is usually the reverse primer that works. Cloning and sequencing is pretty straight forward but time consuming.

I have used methprimer for BSP primer design I have to say it was rather lousy and I pick by eye these days. I am not too sure if the authors have changed the parameters for BSP primers.

Good luck!

Nick

-methylnick-

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Dear Nick,

Is a water bath sonicator good for shearing DNA in a CHIP assay??. I use "SUPER RK 103H from Sch�t labortechnik, i sonicate DNA at 100 strenght for 2 mins + 2 mins in ice cold water (i place the samples on ice after the first 2 mins). The DNA smear on the gel looks the same with sonication time points of 1.5+1.5 mins or 2+2 mins or 3+3mins. Could you please explain.

Thank you.

sridhar.

-sridhar-

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I am not too sure what you mean by the DNA smear looks the same....what is the approximate size of the smear? I have not tried using a water bath sonicator but I have heard it is better than the the conventional tip sonicator that we routinely use.

are you able to post a image to the board for all to see? From the info you have given, it could mean that the settings you used have completely sheared the DNA to completion or depending on the size, not sheared the chromatin at all.

Nick

-methylnick-

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Dear Nick,

The gel image is in the attachment, consider the tabular column on the left page and the gel images on the right page, ignore the rest. I used "1 Kb Plus DNA ladder" but the marker is not clearly visible. The smear in "lane 9" starts exactly at 100 and ends at 1000pb, this is shearing at 100 strenght for 1.5 mins with a break for 1 min on ice and again sonicating for 1.5 mins( i make sure that the water in the water bath sonicator is ice cold). However there is no big difference between all the lanes.

I used rat cortex cells from Embroynic day 18 for the sonication standardizing expts but i try the same parameters on mice cortex cells from Embryonic day 14, do think that the shearing efficiency is the same? please note that E14 has Euchromatin and E18 has heterochromatin.

Please reply back.

sridhar.

-sridhar-

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hi sridhar,

this confirms my understanding that water bath sonicators are more efficient than the tip sonicators.

You want to see a smear ranging from 100-1000bp after sonication. I can't see your ladder however, it seems you have fully sonicated your chromatin with the shortest condition, there are some high molecular weight stuff but as you can see the majority of the smear is within the right size range, have these been RNAse treated? it seems you have stackloads of DNA but this could be because you are also seeing RNA.

it looks fine, I think I will ask my PI to get a waterbath sonicator!

Nick

-methylnick-

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hi nick,

the samples in my gel image are RNAse treated. I have bands in the PCR reaction for the"no Ab" control of my CHIP, however these bands have a low intensity when compared to the +ve controls. Will the tratment of "agarose beads" with BSA completely remove the bands in my PCR reaction?? can you commet about that??

Is there an alternative method to purify DNA other than Phenol: chloroform extraction before proceeding for a PCR in a CHIP assay?

sridhar.

-sridhar-

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Dear nick,

I have a important question to ask you, my sonicated products were run on a 0.8% gel at a very low voltage for about 1.5 hrs which gives me a thick smear between 200bp to 1.2kb, when i run the same samples at a very high voltage, i have a very thin smear all throughout the gel, does it mean that my samples are properly sheared or not, please reply this back.

sridhar.

-sridhar-

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running at high voltage and getting that result doesn't really matter your size range is fine for IP

N

-methylnick-

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Greetings fellow MSP and BSP'ers!

All of your postings on this board have been most helpful! And I thank you all for that.

I understand the concept between the differences in the primers for MSP vs BSP. However, if you're gonna do PCR and get a band regardless, then can't you sequence the band in MSP too? Or has the DNA been treated differently in MSP than BSP so that sequencing cannot be performed in MSP?

Thanks

-purplefetus-

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Do you have any recommendations as to what company has the easiest to use kit for methylation studies?

Thanks

-purplefetus-

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QUOTE

However, if you're gonna do PCR and get a band regardless, then can't you sequence the band in MSP too? Or has the DNA been treated differently in MSP than BSP so that sequencing cannot be performed in MSP?

The modification step is the same for MSP and BSP. If you want to do sequencing, only sequence BSP products. Sequencing MSP products doesn't make sense.

From its protocol, it seems to me EZ DNA Methylation Kit is very simple but I have not used it myself.

-pcrman-

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QUOTE(pcrman @ Mar 1 2006, 09:23 PM) [snapback]42731[/snapback]

QUOTE

However, if you're gonna do PCR and get a band regardless, then can't you sequence the band in MSP too? Or has the DNA been treated differently in MSP than BSP so that sequencing cannot be performed in MSP?

The modification step is the same for MSP and BSP. If you want to do sequencing, only sequence BSP products. Sequencing MSP products doesn't make sense.

But aren't MSP studies not as credible as BSP studies? Sequencing your bands will give THE most conclusive evidence? no? That's why I was wondering about sequencing MSP products.

-purplefetus-

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MSP tests only CpGs within the primer set, BSP tests all CpGs within the amplicon (a whole lot more potentially) so I wouldn't say BSP is more crdeible, but that BSP assays more sites and MSP, MSP is a quick way of determining methylation status without the need to goto sequencing, and you shouldn't be sequencing your MSP products as these results, as you said, are meaningless.

Nick

-methylnick-

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