Bisulfite sequencing - why cloning? - (Jun/13/2007 )

Hey everyone!

I am planning on doing bisulfite sequencing in addition to the MSP results I had gotten previously, in order to get a broader knowledge over the whole promoter region (and not just the areas I had chosen for the MSP experiments).

So, when I was gathering information about the bisulfite sequencing, I noticed that most people clone the amplified sequence into a vector (like TOPO) before sequencing.
Is there a reason, why this approach is preferred over the direct sequencing of the PCR product? Is this because of a potential difficulty in designing the 2nd sets of primers for the nested PCR?

Also, since my region of interest spans ~1200 bp - do you think it will be a problem for the sequencing, because of the high occurence of "A" in the (cloned in) PCR product?

Thanks for your help!

Hazel

Hi HAzel,

cloning and sequencing is performed so that you can look at individual methylation patterns from essentially one allele of one cell. To get a good indication of what all your cells are doing, you would need to sequence many.

TOPO of pGEM cloning is used because it is easier to clone into a vector if you were using Taq in your PCR, that adds A to the products.

1200bp can be done, but you need to perform two sequencing runs on the one clone because you can get only upto 700-800bp of good phred20 sequence. So it would be better to design two primer sets for this region to cover the whole thing.

Nick

Hey Nick,

thanks for your reply. I'm not 100% sure I completely understood. So please correct me if I'm wrong:
From your answer I understand that by cloning the PCR product into a vector and subsequent colony picking/miniprep/sequencing, I get the information from one PCR product (I guess there could be many, due to different cells and incomplete bisulfite conversion).
However, if I would just sequence whatever I got from my PCR run in the first place, I would get many different sequencing results, which could be very confusing.

Is that what you mean?

Also, would you use primers which are designed by MethPrimer for the "internal" region, since TOPO has its own sequncing primers (T7, T3 or M13)?

Thanks for your help!

Hazel

Hazel, you are on the right track, one clone insert = information from one cell and one allele but you don't know which. Direct sequencing gives you an average over all the cells you had in the starting sample.

as for primers for sequencing, BSP primers are lousy sequencing primers, so if you clone into a sequencing vector, it is better to use a sequencing primer such as T7, Sp6 or even M13.

Nick