BSP, MSP - many different ways - (May/19/2006 )

Dear all,
I am just new here and hope to get some useful advices:

For BSP
1. Do I need (or is it obligatory) to cut up genomic DNA before bisulf.convertion by restr. enzymes, or not?
2. Which software should I use for BSP primer design? Is MethPrimer works well?
3. Is it obligatory to do nested PCR?
4. Wich polymerase to ise for amplification?
5. Which kind of + controls one can have?

For MSP
1. Do I need (or is it obligatory) to cut up genomic DNA before bisulf.convertion by restr. enzymes, or not?
2. Which software should I use for BSP primer design? Is MethPrimer works well?

Thank you in advance, and...hope to get assistance from profis

For BSP

1. It is necessary, you need to reduce the complexity of your DNA to ensure proper denaturation into single strands for efficient bisulfite conversion. Pick enzymes that do not digest your region of interest or run your DNA through a syringe to shear it.
2. People use methprimer for primer design and I have to say from experience, people get mixed results. I would suggest using Perlprimer as the primer design parameters are better for BSP.
3. Not necessary, but it's preferred, you have very little starting template for your PCR and you may be lucky to obtain a product that can be seen on a gel after 30 cycles. However, if you don't then a second round of PCR is necessary and in order to increase specificity, nested primers would be better, you can use the same primers in the second round as your first, but this is not favourable as in normal PCR.
4. Again it's a matter of choice, it is not necessary to use a proof reading Taq, normal Taq is fine, people also use hot start Taq, but it's up to you.
5. Positive controls would be to look at sites of known methylatioon status, or to artificially methylate some DNA with SssI methylase and then treat this.

For MSP

1. same as above
2. MEthprimer is the only software I am aware of that is free and designs MSP primers.

Good luck!

Nick

4. I've had success using Amplitaq Gold. I amplify my BSP products over 45 cycles! The beauty of Amplitaq Gold is that it is an inactive polymerase. However, every time it reaches ~94 degree C more of the polymerase becomes activated. So it can go the distance when you need to amplify >35 cycles on PCR.

Hope it works out for you.

Isn't it possible to use Perlprimer to design BSP primers nested and otherwise. They usually give several primer pairs in their output. Is it possible to select 2 pairs from then results generated, 1 that is larger, ~1kb and the other around 200bp that fall within the larger amplicon?