positive control for BSP? - (Mar/24/2009 )

Can i use Universal methylated DNA as positive control in BSP?

I got a problem. I started doing BSP recently. After PCR, my band should be of 240 bp. But i am getting one clean band around 280 - 290 bp in all of my samples. Even the universal methylated DNA also showed only that band with 280-290 bp.
But one of my sample showed two bands, one brighter band at 280-290 and other faint one at 250 .

What should i do?
I am thinking to send one 280 bp band for sequencing.

need advice,
Thanks

You can use Universal methylated DNA as positive control because it is supposed to be fully bisulfite converted. BSP primers only differentiate bisulfite converted from unconverted DNA regardless of methylation status.

Regarding the product size problem, first make sure the DNA template does not contain polymorphic sequence. Most likely, it is caused by non-specific priming because bisulfite conversion reduces DNA complexity and increase non-specific binding of BSP primers. Sequencing the product will tell you what happened.

Thanks PCR Man for replying.I will send the bands for sequencing.
Just for now,
So as i am getting only clean 280 bp band in positive control (methylated bisulfite converted DNA),Not the 250 bp band; so theoritically do you think that is actually my band and somehow in gel it is showing up as 280 bp instead of 240 bp (that should be my product size). I mean, before i get sequencing result, should i concentrate on 280 bp band ?

Thanks again.

pcrman on Mar 24 2009, 08:37 AM said:

Sometimes if a gel is not run long enough the indicated band size may not be accurate. Try running your gel longer to see if the band moves to 250 bp.

Is the band sharp and bright? In the mean time you can raise annealing temperature a bit to see if you can get something different.

pcrman on Mar 24 2009, 09:21 AM said:

thanks . i did run long, it is still 280 bp.
and the band is really sharp and bright in all samples. The 250 bp band is faint and some kind of smeary.
Yeah, today i raised the a. temp by 2 degree to see what happens.

Thanks.

I would like to put my two cents in and ask if you know if your gene of interest has a pseudogene? you could be amplifying off that.

Also, you would be very unlikely, but it is remotely possible, that your priming sequences are similar to another region of the genome (after bisulphite conversion) hence the different sized band.

nick

methylnick on Mar 24 2009, 01:01 PM said:

Its fibronectin 1 and i am really struggling with it. I dont have any idea whether it has pseudo gene.

After increasing the A. temp (from 47.5) by 2 degree (49.5 degree), i got same 280 bp band. For a chance, i increased a little bit more (51 degree) (actually the melting Temp predicted by IDT is around 48 but by Primer 3, its 53, so trying), let's see what happens

now, i guess if i get similar band here, i will send that to sequence.

Thanks

hmmm one way to find out would be to blast the unconverted primer sequences, or even use PCR on Genome Browser.

if you get multiple hits then there would be your problem.

nick

Try bisearch, it allows you to blast bisulfite modified genomic sequence.

Thanks for replying me. After increasing the A.Temp to 51 degree, it is still giving sharp bright band at 280 in all of my sample. I am sending it for sequencing.

I did primer blast, No hit.

I tried Bisearch. This is the first time i used it. The result is:

PCR product(s) on the bisulfite transformed sense chain:
No PCR product should be generated

PCR product(s) on the bisulfite transformed antisense chain:
1. Chromosome 2 (len: 240)


and Fibronectin 1 is also in Chromosome 2 and PCR product with my primers should be 240 bp.

I noticed another thing in Bisearch. When i tried to see score of my primer, the result is "The score is above the upper limit (i.e. very wrong primer pair)". WHat does it mean?

Thanks a lot.