Setting up bisulfite sequencing in lab - (Apr/10/2014 )

Hi Phage434,

Thanks for your quick reply. I am now clear on the biotinylated issue!

I guess what still confuses me is:

- bisulfite specific primers: design according to certain criteria, forward and reverse.that part I think I understand OK.

- sequencing cycle: we use only one primer - forward or reverrse? Are these primers different from our bisulfite ones? This is the part I don't fully get.

Thank you!

For Sanger sequencing, it is common to perform two sequencing reactions on the same sample, one with a forward primer and a second with a reverse primer. This is done to catch potential sequencing issues. These primers can be the same as the PCR primers.

For next generation sequencing, it can be confusing, because often there is a requirement that very specific sequences be present at each end of the sequenced molecule. These are often added by adapter ligation, but can also be added as 5' extensions to a PCR primer. You really need to know which machine is being targeted, and it is a whole subject in and of itself.

Ok great, I get it. We are using Sanger seq., therefore will use both directions. After one reaction we carry out the following, with or without some purifying before the second?

Oops sorry sent too fast - just eanted to say thanks again!!

Typically the two Sanger sequencing reactions are carried out at the same time. After splitting the purified PCR product, each primer is added, and the sequencing reaction performed.

Both are not strictly necessary, depending on how precisely and reliably the results are needed.

Thanks for your invaluable help!

once I have sequenced in both directions separately I then realign each sequence (forward on one hand, reverse on the other) with the original sequence or amongst themselves? that part confuses me :$

Each forward/reverse sequence for the converted and unconverted DNA should align and (accounting for the reverse complement) be identical. As you gain more confidence in your sequencing ability, you may eliminate the reverse sequence, but for now I would do it.  The converted and unconverted should align except for the C's (or G's if you made primers for the reverse strand). C's that are present in the converted sequence started out as methylated. C's in the original sequence that appear as T's in the converted sequence were not methylated.

Ok thank you very much! I think I'm having problems just to understand EXACTLY how the primers work - I want to understand everything perfectly! Will continuento read through the web to get the full picture, if you know of any reference which is a good read I would appreciate it!!! Thanks for all the help, it's verybappreciated!

An important starting point, if you really want to understand what is happening, is to realize that following conversion, the two strands of DNA which started out as reverse complements, are no longer complementary. The upper strand will have most of the C's replaced with T's. This also happens to the lower strand, but the sequence is reverse complemented, so when you look at it as normally written, most of the G's are converted to A's.

This means that when you are designing priimers, you are designing primers pairs for either the top or bottom strand, and those primer pairs are different. For the top strand, your forward primer will have mostly G's, and the reverse primer mostly C's. For the bottom strand, your forward primer will have mostly C's, and your reverse primer mostly G's.

ok, so:

- when I use programs to design primers (i.e. Zymmo Research, etc..) the primer pair given is used for both the PCR following bisulfite conversion & the PCR cycle sequencing. The forward primer is for the top strand, and the reverse for the bottom strand?

- in theory, with only this primer pair I would have enough?

Thanks!