MSP primer design help - (Jun/02/2006 )

I am working on bisulfite sequencing of the promoter region of a gene. I'm trying to do this by divvying up the region(the entire gc rich region is about 500 bp) in smaller segments. I am new to the field and have been at this for about 5 months.......with no success. I have designed about 20 pairs of primers. After many failures, I finally thought I hit on the right pair. The region amplified is supposed to be 115bp and I do see a band(though it is fuzzy).

However, after trying to ligate and transform without success several times we are starting to doubt if this is really double stranded in the first place. I have also tried direct sequencing without success.

I do use the Quiaquick column to clean up and about 80% of the time do see my band after clean up.

One thing is I use very high conc of primers(2 micro molar) in my pcr reaction. Could my band be just primer dimer?

Would anyone have the time to look over my primers for my gene sequence to give me ideas? I used the primo software to design them. I'd be glad to email them to you........ I also tried using the MethPrimer site, it identifies my CpG rich region but will not let me design primers in that region.

"CpG island prediction results
(Criteria used: Island size > 100, GC Percent > 50.0, Obs/Exp > 0.6)
1 CpG island(s) were found in your sequence
Size (Start - End)
Island 1 539 bp (1235 - 1773)
Primer picking results for bisulfite sequencing (or restriction) PCR
No primers found!
"
Why would it give me this message?

Please help!

Here is my gene sequence. The promoter region is the gc rich region immediately upstream of the "atg" in small case.
TGCTCTGTTGTCCAGGCTGGAGTGCAGTAGCGCGATCTCGGCTCACTGCGAGCTCCGCCTCCCGGGCTCACGCCATTCTC
CTGCCTCAGCCTCCTGAGCAGCTGGGACTACAGGCACCCACCATCACGCTCGGCTAATTTTTTTTTTGTATTTTTAGTAGA
GACGAGGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCTGCCTCGGCCTCCTAAAATGTTG
GGATTACAGGCATGAGCCACCACGCCCAGTACCCGCTTCTGAATATTTTAAGAAGGGCATCTTGCCAGTGGACTCTAGTCC
CAACTTAGGTCTCAACACAGCTAGGAGGGGAGATGTGTGACTGTTCTATTTTGTTTGTTGGCTAGTCTGACTTAAATACTT
ATTAGTATTAGTATTGTTATTATTTTTTGAGACAGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGGTGCAATCGTGGCT
CACTGCAGCCTCGACCTCTTGGGATCAAGCGATCTTCCCACCTCAGCCTCCTGAGTAGCTGGGACTACAGGCATGCGCCTG
GAATATTTTTGTATTTCTTTTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGAAGGTCTCGAACTCTTGAGCTTAAGT
GATATGCCTACCTCGGCCTCCCCAAGAGCTGGCATTACAGGCTTGAGTCACCACTCCCAGCCTGAAGCATTATTATTATTA
TTATTATTATTATTATTATTATTATTATTAGAGACAGAGTCTCACTGTGTCACCCAGGCTGGAGTGCAGTGGCATGATCTC
GGCTCACTGGGACCTCTGCCGCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGCACCTG
CCACTGCGCCCGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATCTTGGCCAGGCTGGTCTTGAACTCCGACC
TCAGGTGTTCCACGTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGCACCCAGCCAAAACACTATTAT
TAAAATCACTTATTTGTAAACAGGTAATATTTGCACATGATAAGTACAAATCATTTAGTAAAAAGTCAACGGTGACAGGCT
TGCTTTGTTTTATTCCCTTTCGGCAAACAGGTGCCCATTTCCTCGCATTTTGTCTATGGTGCCTTTCTTGCAACAAAGGCA
GAGATGAATAGCTGCTACAGAGACTCAGGGCCTACATATCAGAGAACATTTTCTATTAGGCCCTTTACAGAAACCTTCGCT
GATTTTTTTTTTTTAGTATTTTACCATTTCAGACAATGTTGCTATGCTATCTTTGTATATCTGTCATTTTGCACATGGGAG
TTTCTGTAGAATGATCTCTCTATGTGGAAATGCTAGGTCTTAAGGTGTCAGCATTTGTCGTTTTGATAGGTGTACCAAATA
GCCCTCTAGGGAAGTTGAACTAATTTATACTGTCTTCAGCGAAGCATGCTGGACCTGTTCCCCACAGCCTCCACTAACCTT
GTTAACATCTTGATACTTGCCGATCCGAAAGGGAAACACGATGCTCATTGTAGTTTTAATTTGCATCTATCTGATTATGAA
TAAAGATTAGCATCTTTTCAAGTCTAAAAATCTTAAAAACAAAACTCCTGAACTTTTCCCGCCCACAGACTCAAGACTCGT
GACCCGGCCGTTGGCACGACGCGGGACGCCGGTGTGGCAGTGGGGAAGAGGCAGATATCGCGGACCACCCCCGCGCCCCCC
AATTCCTTTCAACCA:ATTCCCCCCAGCCGCGGAGCCCCGCCCCCAGTCCGCCTCTGGCCAGCTTGGGCGGAGCGCACGGC
AGTGGGAGGTGCTGAGCCGCCTGATTTATTCCGGTCCCAGAGGAGAAGGCGCCAGAACCCGCGGGGTCTGAGCAGCCCAGC
GTGCCCATTCCAGCGCCCGCGTCCCCGCAGCATGatgccgcgcccccgcctgctggccgcgctgtgcggcgcgctgctctg
cgcccccagcctcctcgtcgccctggGTGAGTGGATCGCGCCGCCCTCTGCCCCTCGCCCCTCCTCCCCGCGCCGCTCGGA
AGTTTGCCCGGCGCCCGCCCTCCACCTCCACTGTTGACAAACTTAGACAAAGCCCCGGGGACCGGGCTGGGCAGAGGGGCG
GCTTCTTCCGCTGCGCCCTGGCGGGACAGGGGGATGCGGCCCTGCTGTCTCTGCGCTGGGGCTTTTGGGCTGGGACTC*


Here is my primer set
Met 10F Forward 6 66 TATAGATTTAAGATTCGTGATTCGG 116
Met 12R Reverse 122 64 ATTAATTAAAAAAAATTAAAAAACGCGA


CCCGCCCACAGACTCAAGACTCGTGACCCGGCCGTTGGCACGACGCGGGACGCCGGTGTGGCAGTGGGGAAGAGGCAGAT
ATCGCGGACCACCCCCGCGCCCCCCAATTCCTTTCAACCAATTCCCCCCAGCCGCGGAGCCCCGCCCCCAGTCCGCCTCTG
GCCAGCTTGGGCGGAGCGCACGGCAGTGGGAGGTGCTGAGCCGCCTGATTTATTCCGGTCCCAGAGGAGAAGGCGCCAGAA
CCCGCGGGGTCTGAGCAGCCCAGCGTGCCCATTCCAGCGCCCGCGTCCCCGCAGCATGatgccgcgcccccgcctgctggc
cgcgctgtgcggcgcgctgctctgcgcccccagcctcctcgtcgccctggGTGAGTGGATCGCGCCGCCCTCTGCCCCTCG
CCCCTCCTCCCCGCGCCGCTCGGAAGTTTGCCCGGCGCCCGCCCTCCACCTCCACTGTTGACAAACTTAGACAAAGCCCCG
GGGACCGGGCTGGGCAGAGGGGCGGCTTCTTCCGCTGCGCCCTGGCGGGACAGGGGGATGCGGCCCTGCTGTCTCTGCGCT
GGGGCTTTTGGGCTGGGACTC*

The above is the sequence I plugged in to design my primers on Primo.

hi nevergiveup,

that is a great attitude to have, never giving up!

The primers you have picked aren't efficiently selecting for bisulfite converted DNA molecules. Furthermore, 2uM primers in your PCR is way way too much!



The most sequence specific part of your primer is the 3' end. Thus pick your primers such that they end in T's that were C;s before bisulfite conversion (in the sense strand), the antisense would end in A's. From this the primer should have equivalent base composition before conversion, thus there will be a bias towards more T's in your bisulfite primer pair. I typically pick 30mers and hemi nested primer sets to perform two rounds of PCR.

Have another go at ppicking your primers, and PM me if you want extra help.

Nick

Thanks Methylnick. Will design new primers as you have advised. Hope it works but in any case, appreciate ur taking the time!

Hello there,
I came up with these. Any imput is welcome. Please see attachment.

nevergiveup,

I would be inclined to give MethylPrimer Express a shot at picking your primers. Methprimer has some issues I have to say, one of these is your forward primer there are very little conversion events the primer is targeting there.

Good luck!

Nick

hi nevergiveup,
I think your Met 12R Reverse primer is not good. It possess too low complexity. Try to select primer rich in C and T (G and A in sense strain).