Need for Nested PCR - Primers Designed with MethPrimer - (Jun/08/2006 )

Part 1:

Is there a need for nested PCR if i design my primers with MethPrimer? No CpGs are within the primer sequence.

Im going to use a genomic DNA extraction kit followed by the EZ methylation kit from zymo research.

I plan to amplify a 481bp fragment with 33 CpGs and directly sequence the pcr fragment following gel extraction.

Has anyone tried this with success? Thanks for the help.

Part 2:

For primer design has anyone tried to design the primers with the assumption of C conversion to T? If I design my primers in an area not of interest, why cant I just assume C conversion to T and change the sequence of the primers accordingly?

For example...

5' GAGGCCAAAGGCTGAACCCAATG 3'
3' CTCCGGTTT CCGACTTGGG TTAC 5'

Followed by Treatment with bisulfite we have

5' GAGGUUAAAGGUTGAAUUUAATG 3'
3' UTUUGGTTT UUGAUTTGGGTT AU 5'

Now to desing the top primer should I try to use the converted sequence as the template, if I dont care about the sequence change

So my 5' primer would be

5' AAAACCAAAAACTAAACCCAATA 3'

If the melting points of my primers are within 5 degrees would this be a good strategy?

Thanks, Kevin.

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Hi,

in most cases you can assume non CpG C conversion to T, that is the whole crux of the procedure. However you should be cautioned as to which model organism you are using in your studies. With plant's it is a little bit complicated as there are non-CpG methylation with mammalian systems it is safe to say all non-CpG C's are unmethylated and will be converted to T's after bisulfite.

A nested strategy will help as you are starting with a minimal template after bisulfite conversion as it is such a harsh process.

Nick

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Thanks for the quick reply Nick. Its a mammalian cell line.

Any thoughts on my strategy for the primer design? Forget about sequence conservation...convert the antisense strand, and then make the sense strand primer accordingly?

Dose this make sense? Or am I missing something? I havent seen this done...It would negate the primer template missmatching, and only amplify converted sequences? I dont understandy why this strategy isnt used?

Thanks for your help...Im the only one in the lab working on this, and the phd student that is involved with my project dosent care much...lol

Kevin.

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That is how it's been done all the time, otherwise I think i am missing something!

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For this sequence

5' GAGGCCAAAGGCTGAACCCAATGGT 3'

methprimer makes this forward primer

5' GAGGTTAAAGGTTGAATTTAATGGT 3'

Just dosent make sense to me?

This creates mismatches...what am i missing?

Kevin.

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Hi Glassman
This forward primer is correct. However, it can't anneal and extend until the reverse primer comes through to create the complementary template. Remember that the two primers are specific for one strand only. The original two strands will be non-complementary after bisulphite conversion so the other strand won't amplify.

The forward primer you designed in your June 9 post is also correct but is for the other strand. The sequence for the reverse primers in both scenarios will be different because of the non-complimentarity of the +/- strands. Try and draw out the two scenarios on a piece of paper and the "penny will drop". I had to do this to finally get my head around how it all works!

You should be able to get your melting temperatures to within a degree or so of each other.

Hope this helps. All the best.

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Thanks for the help guys...I should be able to design the perimers now...

Thanks again.

Kevin.

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