no methylation vs full methylation - (Aug/20/2010 )

I ran into a problem with I did the methylation test. I am working on a fungus, neurospora crassa. With a ristriction enzyme-PCR method I detected a rigion with light methylation, which is about 10-20%. However, by using the bisulfite conversion followed by PCR-cloning, what I detected is that for those clones (about 20), each sequence either contains no methylated C or has all the C remain unconverted. This result has happened several times. So I am wondering if anybody has any idea about this? I am testing the possibiiity of contamination and see nothing come out from water. Plus I didn't find any contamination so far from my negative (a gene have no methylation) or positive control (heavily methylated >50%), where I could see typical scenario of methylation of positive control which showed a mosaic pattern.
Since the detected methylation is too weird (all C is methylated), my project is stuck here and I don't know how to continue. Thanks a lot for your advices.

sounds like you have PCR bias induced by the primer design

how have you selected your primer sequences?

I selected the regions of bottom strand containing most G but have few C for primer design. I usually do a nesting PCR for one target gene/sequence as one round of PCR never gave a detectable band.
I had thought of the problem of primer design. I appreciate if you can give me a better suggestion.
here is an example:
ncu2266r: TGT ATT GYT ATG GTG TTG TGG GTA AG
NCU2266f: CAT TCA ART CAA RCT CRT ACC CAC ATC

aagctcgtacccacatccccacaggttccagagtttggccggacaaccagtacgggtgcccgaggcgtcctgatg base pairs
ttcgagcatgggtgtaggggtgtccaaggtctcaaaccggcctgttggtcatgcccacgggctccgcaggactac 601 to 675







ccgctgcaagaccgatgacgctgcaaaattgagatctggaggctcaagatactttcggtcgaagccccaccgccg base pairs
ggcgacgttctggctactgcgacgttttaactctagacctccgagttctatgaaagccagcttcggggtggcggc 676 to 750







cgacctcctgtgccacctttccgccaggctgttgtggaaagtcactgcagcgcaccccctttgcgagcaatgacg base pairs
gctggaggacacggtggaaaggcggtccgacaacacctttcagtgacgtcgcgtgggggaaacgctcgttactgc 751 to 825







ttagaagatgtgggacgttgctcgataggcccaccccgcgtggcgtcaaagtggagccgcccacaatcctgagcg base pairs
aatcttctacaccctgcaacgagctatccgggtggggcgcaccgcagtttcacctcggcgggtgttaggactcgc 826 to 900






DpnII
cagcctgtgatcctgataaccgacaagatgggtgcgaatggaggcattttggggccagcaactgcgctcggagtt base pairs
gtcggacactaggactattggctgttctacccacgcttacctccgtaaaaccccggtcgttgacgcgagcctcaa 901 to 975






tccgtccctcacatttttccttggattgcttgcgagtcaggcgcacgtgctctctcgctcggtcattgattgatg base pairs
aggcagggagtgtaaaaaggaacctaacgaacgctcagtccgcgtgcacgagagagcgagccagtaactaactac 976 to 1050







cggattgccggcgaccatgctgattgattgattgattatgctttggagcgaaccatccggattgtgctaggtagt base pairs
gcctaacggccgctggtacgactaactaactaactaatacgaaacctcgcttggtaggcctaacacgatccatca 1051 to 1125







gtaagggatttccagacgaggttatgaccgttgcgacgggcggggttgcgttgaaaatctggttacgaggtcgtc base pairs
cattccctaaaggtctgctccaatactggcaacgctgcccgccccaacgcaacttttagaccaatgctccagcag 1126 to 1200







gctcctggttcagagttgccaaagagtgtgagaggccaagtgagctgctggtaggtaggaaactgcattcatgag base pairs
cgaggaccaagtctcaacggtttctcacactctccggttcactcgacgaccatccatcctttgacgtaagtactc 1201 to 1275







gtggagacgacaagcacagatttagacttacccacaacaccatagcaatacatggtgggttaagaggatttcaaa base pairs
cacctctgctgttcgtgtctaaatctgaatgggtgttgtggtatcgttatgtaccacccaattctcctaaagttt 1276 to 1350

methylnick on Sat Aug 21 01:35:16 2010 said:

sorry laver, I misread your first post,

within each sample, how many clones show complete methylation and how many show no methylation out of your 20? the ratio you see with the clones could reflect your restriction enzyme result but in reality you would need 100 clones to see your 10-20% methylation from your digest result.