Pls check my bisulphite sequencing primers - (Jun/30/2005 )

I performed the bisulphite treatment and amplified the genomic dna by these primers with two rounds of PCR, as described in the attached file, but either wrong size fragment or no bands was observed. I am not sure if its problem of quality of dna, primer design or pcr conditions. could you pls help me to check if the primers are ok or not. thanks

samantha

Hi samantha,

It looks like you are wanting to performe bisulfite seqeuncing?

With the seqeunces you have posted, have they already been "mock" bisulfite treated? Meaning all non-CpC C's are converted to T's? It looks as though it is but I don't think the 3' end of your primers are targeting converted C's just by looking at your forwards, they do not end in a T or a string of T's, your reverse primers do end in A's which is good, but I am not too sure if these are to converted C's to T's or are they to ordinary T's?

To distinguish between a native T and a T that was converted from a unmethylated C, I normally bold and make the font slighty bigger on the T's that were converted from C's, then I will know where all the bisulfite conversions have taken place.

Nick

Thanks for your reply. The sequences I posted are the converted sequences for bisulphite sequencing. Are the primers ok? if not, what should i do or any other factors that I should take into considerations?
Thanks.

Samantha

Here are the original sequences. If you have problems to view the files, pls feel free to say it.

Samantha

Hi Samantha,

thanks for reposting the sequences.

it seems there are a number of primers that seem to me, to be suboptimal.

I would pick the primer seqeunces such that they end is one or more capital T's (that is to say one or more C's that have been converted to T's after bisulfite) that way, you are only amplifying converted templates.

in your D file: the orange primer set are not ideal for bisulfite PCR the F primer doesn't contain any T's and thus will amplify both normal and bisulfite converted templates. The green F primer looks okay.

similarly with the nested.

for the S document, I think your Pink and orange R primers is okay The forwards are also not ideal. The purple primer set is not ideal either.

it is important that the 3' end of each primer ends in an conversion event!!!


have another go at picking the primers.


good luck

nick,

i tried to repick the primers which you think are not fine for bisulphite sequencing. i have reposted the edited version of the files here. Those primers which are on ITALIC are the edited ones. Please give me some adivice on the primer design if possible. Thanks a lot.

Samantha,

that''s better the primers you picked looked good! make sure that your primer pairs have equivalent Tm's and when you perform the PCR set the annealing temp 2C below the calculated Tm, you should get some specific products and they should be mostly converted!

Good luck!

Nick

Thank you so much, nick!

Samantha

Kinda curious. What is the lowest Tms that you normally are willing to go with on bisulfite sequencing primers and what are the normal amplification settings that you use?

Nick, after editing ther primer sequences, i could amplify the fragments with the right size. its a good start! now, i am waiting for doing sequencing. Thank you so much!!!!!!!!!!!!

Samantha