Bisulfite sequencing - (Aug/17/2010 )

I would like to correlate change in promotor DNA methylation and a change in gene expression.
I have a lot of problem with my PCR amplification after bisulfite treatment.
The DNA region of theoretical CpG island is :
GCCAGGCTCTTCCCCCCGCCCCCGGAATCCCCCTCCAGTCCCGCTGCTC
GCAGCCTCAGTCCCGTCCTTGTCAGCCTCGCCTGGTCCTCCCTGCGGGCTAACCCTGTAT
TGCCTGTTGCACTCACCGCTCTCCGGGGTTTGTGACGCCTCTTCTGGGCCCCGGGGACCT
TCCTGGAGGAGCCTGGCCGTGCCAGCTCAAGCTGGAGGTGGGACATGAGGCAAATGGAAA
AGGCGAAGGACGAAGCCCCTCTCTGCATCTGCCCGTGGCCCTCCCAGCCGCAGACTCGAG
CCCGGATTTCAGGACACCCAGAGGACCCGACTCCGGTCTGGATGGGTTTTTCCGCTAAGC
AGGACAGATTGGCAAAGCGCTGGGGCTCGCCGTGGGCCTGAGGATGCAGAGAAGCTGGCG
GGGGAGAGGGGCGGCGGGGCGCGGCCGTCACTCACGCAGGCCGCCACATCCCTCCCC
GCGGCCGCCAGCAGCTCCAGGATCGGGCCGGCGCCGGGGAGCAGCTCCGAGCGGTGCAGGCCG
CTGAGCACTGCCTGGGCGTCCGCGATGCCCCGGGCCACGTGGCGGAGCCGAGCGCAGGAC
TGCGGGGACGAGAGGGCGTTAGAGCGGGCCGCGCCCGGGCCATGCCTCTCCCGCCCACTC
CCGGGCCTCACCGATGGCCGCGGAGGATCCCTCCTGGGGCGGAAGGAGCAGTTGCGCTGC
CCCCAGCTCAGCGCCTCTTCCTCCTGCGGGACAAGCGGCGCTTATCGCATACGGCTAGGC
CCCCTCGCCAGGGCCCCTAACCTCTGCACAGTCTGGGATTCCTGGACGTGGATGGGTACT
GGCAGCGCACGGTCGTGCCTGTCGTGTACTGAACCAGGGAGCTCCCCGAAGGCGCGAACC
AGGGTTGAATTGCACTCCGCGCTCCCCCAGCAAAGCCCCTCGCCCCGACCTGGAGCCGAG
TCCTCCCGGCAGGGCTCCCTTCTGTGATTGACCCTGAGCCTGCGTTCGCGCTGACGACGG
GGACTGCGGGGGTCTCGTGGTGGGAATTGTGGGCGCTGACATAGGAGAGGCGCCTGCTGG
GCGCTAGGACGCAGGACCCCTTGGGACAGGAACGGGTGTATGGGAACCCGGTGGGGCCAG
GGTCCCAGGGGGCACAGGGGCTGGGCGGTGACTTACGTAGCGGTCCCTCAGCGCCTTGGC
AGCCGCCAGCGTCCGGGGCTCCAGCGAGCGGTAGTGCGAGAGCAGGCAGCGCCGGGGGGC
CTTCTGCGATCACCGTGCACAGGACCCACAGCCCCGCGGCCACTGCGGCCCAGACACTCG
GCCGCATCTCTGCTTCTGCAGCAGGCGAGAGACGTCAGGGAAGCCAAAGAGAGGGTCCAG
CGCGTCCAGCCCCCCGCCTTGGGTTAGGATCCCAGGGAAGGCAATGCTCGGAGCGTGAAG
GCACAGCACACACAGTGGGAGAGAGAGTGGGAGCCGGCCCCCTCCTCGCCTTGGCCTCTG
CCCTCACTC
I would like to analyze all this region, but I think it's better to screen it with amplification of small fragments. I have tried to design primers with MethPrimer:
1- left primer TTGTATAGTTTGGGATTTTTGGA
right primer CAAAATCAATCACAAAAAAAA
2- left primer GTTTTAGGGGGTATAGGGGTTG
right primer ACATTACCTTCCCTAAAATCCTAAC
3- left primer TTTTAGGGGGTATAGGGGTTG
right primer CTAAACCCTCTCTTTAACTTCCCTAAC
I obtained amplification but sometimes not or multiple bands. I have tried to extract each band and to purify it and then to clone it in a pGEM vector. I obtained very beautiful sequences but I do not know what these sequence correspond to!!!
Could you help me? Do you think my primers are OK? Can I used another primers to study the methylation pattern of all the island?
Thanks a lot.....

Bib

did you try to marry your sequencing data to converted or unconverted sequences to your region of interest?

Does your region of interest consist of a potential repetitive sequence?

This could be why you are getting what you are getting.

nick

Hi Nick
Thanks a lot for your suggestions.
1- I did a sequence alignment with converted or unconverted sequences, without results.
Moreover I used BiQ Analyzer and I obtained only 35 % of sequence identities....
2- To my knowledge the region of interest does not consist of a potential repetitive sequence but could you give me a database which will allow me to verify?
I don't really understand why this could be an explanation.
Best,

Bib

methylnick on Wed Aug 18 03:03:15 2010 said:

you could try running your query through methblast.
http://medgen.ugent.be/methBLAST/