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about the methylation content (MSP) - (Apr/19/2006 )

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"After you have done a sucssessful PCR, purify your PCR products and use the fresh products to do a TA cloning (ligation->transformation->plating). You will see white and blue colonies on the plates. For each PCR reaction (transformation) or DNA sample, randomly pick ten white, big, shiny and well isolated colonies using a yellow pipet tip and drop the tip into a 14-ml or 50-ml tube containing 2-5 ml LB medium with antibiotics added. Grow the bacteria overnight at 37C with virgorous shaking. The next morning, take out the tubes and take 1 ml overnight growth for miniprep. You can pick more than 10 colonies (such as 12) just in case some minipreps fail. If cost and labor is not a problem, screen 20 colonies. After you got the plasmid DNA, send it out for sequencing. "

Hi , everyone, I am confused that after I get the MSP product (about 150bp), I send it out for sequencing just as the above description. I can only get the methylation information of the selected fragment in the promoter but not that of the whole promoter. Does this reflect the methylation status of the promoter? Is it OK for the paper to be published?
Another question is how to calculate the methylation ratio after sequencing.

I am the first person to do the methylation experiment in the lab. Sometimes the information I unterstood from the published paper is not quite right. So, I am very glad to get your help !
Thanks! smile.gif

Linksys

-linksky-

linksky,

it is not wise to sequence an MSP product as the primers for your MSP are biased to either methylated or unmethylated templates, so if you sequence it, you will get a very biased result.

you should use BSP primers that do not have this bias and then sequence those.

MSP primer sets tend to only amplify short DNA fragments of approximately 150bp, BSP you can go larger and can encompass the entire promoter region.

With MSP methylation or non-methylation is guaged by the presence or absence of a PCR product, it is not necessary to go any further and sequence the product.

hope this helps

Nick

-methylnick-

QUOTE (methylnick @ Apr 19 2006, 08:13 PM)
linksky,

it is not wise to sequence an MSP product as the primers for your MSP are biased to either methylated or unmethylated templates, so if you sequence it, you will get a very biased result.

you should use BSP primers that do not have this bias and then sequence those.

MSP primer sets tend to only amplify short DNA fragments of approximately 150bp, BSP you can go larger and can encompass the entire promoter region.

With MSP methylation or non-methylation is guaged by the presence or absence of a PCR product, it is not necessary to go any further and sequence the product.

hope this helps

Nick


Thanks a lot. If the promoter has been confirmed to be methylated using MSP, I should detect the methylation status using BSP further and then sequencing. It is easier or quicker to get the methylation formation using MSP method first. Thank you again! smile.gif

-linksky-

linksy, it is easier with MSP if you know that the primers in msp tests sites that reflect the methylation status of the promoter.

Usually BSP is done to assy every CpG site within the promoter and with this data MSP primers can be designed for a rapid assay (in MSP all you do is convert DNA and PCR amplify).

For BSP there are a few extra steps to take, for instance sequencing! and this would involve cloning the amplicon and sequencing a number of clones or direct sequencing which is more fiddly.

Nick

-methylnick-

[quote name='methylnick' date='Apr 21 2006, 10:57 PM' post='48888']
linksy, it is easier with MSP if you know that the primers in msp tests sites that reflect the methylation status of the promoter.

Hi, Nick:

Actually I don't know the sites for MSP that can reflect the methylation status of the whole promoter. So I have to consider to detect the whole sequence of the promoter using BSP method. Another consideration is to use methylation specific restriction enzyme. Maybe a relatively methylation content can be got. Comparing these two methods, I prefer to BSP because ligation reaction is rapid and the following procedures are simple. The most important thing is I can get the precise methylation sites from BSP.
The question is I got two bands from MSP in my samples group. One is at the same MW level as those of negative and positive control group (Only one band is detected in the two groups); The other is very close to the previous band (smaller one) which might be the nonspecific band. Is it necessary for me to do nest-PCR?

Thanks for your help!
linksky

-linksky-

Linksky,

there are a lot of little nuggets scattered on this forum with regard to BSP, have a hunt around and good luck with it.

Nested PCR is always a good idea as it increases the specificity of your PCR.

Nick

-methylnick-

QUOTE (methylnick @ Apr 25 2006, 08:05 PM)
Linksky,

there are a lot of little nuggets scattered on this forum with regard to BSP, have a hunt around and good luck with it.

Nested PCR is always a good idea as it increases the specificity of your PCR.

Nick



Thank you so much! Nick.

-linksky-

QUOTE (methylnick @ Apr 21 2006, 10:57 PM)
For BSP there are a few extra steps to take, for instance sequencing! and this would involve cloning the amplicon and sequencing a number of clones or direct sequencing which is more fiddly.

Nick



Hey Nick,
Why do you say direct sequencing is more fiddly? Is that data not as reliable as sequencing via a number of clones?
Thanks

-purplefetus-

purplefetus,

Direct sequecning is a little more fiddly because the fate of the sequencing reaction relies heavily on the primers you use, and with direct sequencing you are using the same primer you used in the bisulfite PCR which in normal PCR's is not an ideal thing to do. With cloning and sequencing, these are sure fire steps to getting a result and you are using tried and tested sepquencing primers such as Sp6, T7 and M13, you have to make a serious mistake to stuff up the results.

Furthermore, direct sequencing gives you a nice overall methylation status of all amplicons in the PCR, with cloning and sequencing you can look at the actual methylation pattern of individual clones and hence individual loci within one cell (for each clone), direct sequencing doesn't have this power.

Nick

-methylnick-

QUOTE (methylnick @ Apr 27 2006, 07:57 PM)
Furthermore, direct sequencing gives you a nice overall methylation status of all amplicons in the PCR, with cloning and sequencing you can look at the actual methylation pattern of individual clones and hence individual loci within one cell (for each clone), direct sequencing doesn't have this power.

Nick


hmmmm, so, overall if I just did direct sequencing of my BSP products would it be highly criticized? ohmy.gif Would those findings not be valid in the scientific community unless cloning and sequencing was performed?
Thanks!

-purplefetus-

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