Problems with Bisulfite PCR - (Jul/08/2008 )

Hi Guys!

I started on a new project involving DNA methylation and MSP PCR recently. I used the zymo research DNA methylation kit for bisulfite conversion. My untreated primer set and template works just fine, but the non-methylated and methylated primer sets are not working. Also I used the untreated primer set as a negative control for treated DNA, but it tests positive, though the bands are faint. I have tried 3 primer sets so far at different annealing temperatures, but have had the same problem everytime. Has anyone used the Zymo methylation kit? Are there any specific things to keep in mind while designing the primers? I read through the forum and will be trying out decreased annealing (55 C) and extension temperatures (68 C) today. There seems to be an abundance of information available here, I would really appreciate some help. Thanks!

Are you going to run qMSP or just regular MSP? I would like to have a look at your sequence and primer sequence if you want

I am planning on running a regular PCR right now, but will probably run a QPCR eventually.

Primer sequence
Forward Untreated 5' GAGCTCCAGGAGTTGTACG
Reverse Untreated 5' TGATGGTGGCTTGGGCACG

Gene sequence
CAAGCACTGCGCCTAGTGATCTCCTTCGGGTTTGAGCCAGTCCATGGGGAGGAGCCGTCCACCAGGCAGCCTCAGGGAGA
GTGGGCGGGTGTCAAGGGAAGCCCACGGCCCCAGCTGGCCCCCTTGTGTCTGAGCCCCCATGGCTTCCTCTGCCTCTAGGC
TATGCCCACTGGAAGGGCCAAGTGCTGAATTCAGATGAGCTCCAGGAGTTGTACGAAGGCCTGAGGCTGAACAACATGAAT
AAATATGACTACGTGCTCACAGGTAGGTGCCGGAGCAAGCTGCCGCAGGGGACTACGCACCCCACTCCAGCCAGGGTGGGC
TCAGGGAGGTCCAGAGCACCCCCGCCCTGGGCCTGTCTCGGGTCCCTGCCCCTTCAAGGAGGGCCTTGGCGTGCCCAAGCC
ACCATCACACCCCCGGTAGCCTGGGGGGTCCTGGCCACACTATGCCTGCCGGGACAATGACTGCCCAGGCTGTG

Really appreciate your help. Thanks!

Do you think the region is worthy of CpG island analysis? It does not look like CpG island-rich region.


caagcactgCGcctagtgatctccttCGggtttgagccagtccatggggaggagcCGtccaccaggcagcctcagggaga
gtgggCGggtgtcaagggaagcccaCGgccccagctggcccccttgtgtctgagcccccatggcttcctctgcctctagg
c
tatgcccactggaagggccaagtgctgaattcagatgagctccaggagttgtaCGaaggcctgaggctgaacaacatgaa
t
aaatatgactaCGtgctcacaggtaggtgcCGgagcaagctgcCGcaggggactaCGcaccccactccagccagggtggg
c
tcagggaggtccagagcaccccCGccctgggcctgtctCGggtccctgccccttcaaggagggccttggCGtgcccaagc
c
accatcacaccccCGgtagcctggggggtcctggccacactatgcctgcCGggacaatgactgcccaggctgtg

I am not planning on analyzing the region. This gene has already been analyzed and characterized as a CGI I need a positive control for my other reactions. A gene which is methylated in normal cells.

primer design is usually the problem with failed PCR reactions especially with MSP.

I don't think zymo has any control primers for you to use to detemine if you have converted DNA after using their kit.

Do you have any primers that you know work on converted DNA? if so, have you tried them on your DNA?

I would do this to ensure you have good converted DNA to start with before playing around with your primer set of interest, because there maybe very little or no template to amplify off anyway!

Nick

Thanks Nick!

Since I am designing primers for MSP for the first time are there any specific things I need to keep in mind? My advisor says that just a single base pair difference at the 3' end will cause the primers to specifically anneal to its complementary sequence. Have you done anything similar? Are there any problems I should anticipate with this design? Also have you amplified any endogenous sequences from normal cells using MSP? I cannot find much in the literature about it. Most of the research seems to be done on cancer cells. I found a couple good papers which have researched DNA methylation in normal cells, but they did BSP, so I cannot use their primers. Do let me know. Just as a side note, this forum is extremely helpful!!!!!

Ciao

you advisor is right about designing BSP primers towards cytosines that have been converted to thymine at the 3' end. One is bare minimum but more at the 3' end the better.

There are notes I had pinned up long ago about primer design rules.

There are programs that pick primers for you and this have also been pinned in the forum.

We have been doing bisulfite PCR and sequencing on "normal" non-cancer cells quite routinely. Best to have controls you know that work.

good luck!!

Nick

Thanks Nick!

I did try some of the primer design software...... but hand picking seems to work better for me. So this is how I designed the primers. F=Forward, R=Reverse, U=Untreated, M=Methylated, N=Non-methylated

FU: CAGATGAGCTCCAGGAGTTGTACG

FM: CAAATAAACTCCAAAAATTATACG

FN: CAAATAAACTCCAAAAATTATACA


RU: GGGTGTGATGGTGGCTTGGGCAC

RM: GGGTGTGATGGTGGTTTGGGTAC

RN: GGGTGTGATGGTGGTTTGGGTAT

Does that seem right to you??????

Thanks!

so you are using these primers to test one CpG (found on the Forward primer at the 3'end?) it is good you are using the same sequences for both methylated and unmethylated primer sets.

it is possible to incorporate more CpG sites within your primers if you wanted to.

You need to make sure they are of the similar Tm's so it is possible to perform PCR of both at the same time.

Nick