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Why should it be digested with a restriction enzyme before treated with bisulfi - (May/15/2007 )

Hi everyone,
I am a rookie here.I have done the MSP several times,but unfortunately I didn't get good results.I always get several other bands beside my objective band. And the modified results aren't always stable. I nearly get frustrated with it .
I also have another question.In most protocols,they refer to a point that you'd better digest your DNA sample with a restriction enzyme before treated with bisulfite. Is it pre-requisite? I didn't do that in my experiment.
Could anyone help me ,please? Thanks.

Amy

-epilab-

The bisulfite reaction works better if you have smaller fragments of DNA. You can also shear your DNA template before the bisulfite reaction by passing it through a 21G needle several times.
If you see full conversion in your sequences, than this step is not necessary. But it doesn4t hurt.

-krümelmonster-

Thanks to krümelmonster,
Is it right any restriction endonuclease may be used as long as there is no its site in my amplicon?
How to measure whether the conversion is full? Is it that my negative control could be amplified with U primers but not with M primers?

Sincerely
Amy



QUOTE (' date= @ post=)
The bisulfite reaction works better if you have smaller fragments of DNA. You can also shear your DNA template before the bisulfite reaction by passing it through a 21G needle several times.
If you see full conversion in your sequences, than this step is not necessary. But it doesn4t hurt.

-epilab-

Hi Amy,

it's true, your enzyme should not cut your region of interest.
There are two ways of checking for full conversion:
1) bisulfite sequencing - no non-CpG-C traces should be visible
2) you can use restriction enzymes cutting at any motif containing non CpG-C's. If the conversion is okay, the Restriction landmark will change accordingly.

You can get a rough picture of what is going on if you try to amplify your region with primers designed for the unmodified DNA (that is not your M primer, as methylation only conserves CpG-C's). If you're able to amplify fragments with this primer after bisulfite modification, the treatment was not effective.

BUT - Most problems occur due to insufficient primer design, not insufficient bisulfite modification! Your problems are also more likely attributablme to the primers or PCR conditions. Please share primer sequences + conditions with us, than we can help you mor efficiently.

Krümel

-krümelmonster-

Hi Krümel,
my methylated (M) and unmethylated(U) primers come from a published paper as followed:

M : −130 GAGAGCGCGTTTTCGTTTGGC −32 CGCCTCCTTTTCACTCCTACG
U : −130 GAGAGTGTGTTTTTGTTTGGT −32 CCACCTCCTTTTCACTCCTACA
The conditions offered from the paper is M (annealing 59℃ cycles 34) and U(annealing 55℃ cycles 34),however,I can't get good results under these conditions,so I change the amend it as M(annealing 61 ℃ cycles 35) and U(annealing 60℃ cycles 35). It results a little better even though not perfect .

I still have another two pairs of primers which haven't been amplicated . Could you be kind to check them for me ,please?
The first pair :
position sequence position sequence annealing(℃) cycles
M −173 TTTTTAGCGAATTTTTACGTAC −22 ATAAAACGACGCGCACCTACCG 55 35
U −165 GAATTTTTATGTATGAGGGAGGT −22 ATAAAACAACACACACCTACCA 55 34

The second pair:
M −234 GTTCGTTTGACGTTAGGAAGTC −34 GCCCAAAACCAACCGCCTACG 55 35
U −234 GTTTGTTTGATGTTAGGAAGTT −34 CACCCAAAACCAACCACCTACA 55 34

Thanks!

Amy


QUOTE (krümelmonster @ May 15 2007, 11:44 PM)
Hi Amy,

it's true, your enzyme should not cut your region of interest.
There are two ways of checking for full conversion:
1) bisulfite sequencing - no non-CpG-C traces should be visible
2) you can use restriction enzymes cutting at any motif containing non CpG-C's. If the conversion is okay, the Restriction landmark will change accordingly.

You can get a rough picture of what is going on if you try to amplify your region with primers designed for the unmodified DNA (that is not your M primer, as methylation only conserves CpG-C's). If you're able to amplify fragments with this primer after bisulfite modification, the treatment was not effective.

BUT - Most problems occur due to insufficient primer design, not insufficient bisulfite modification! Your problems are also more likely attributablme to the primers or PCR conditions. Please share primer sequences + conditions with us, than we can help you mor efficiently.

Krümel

-epilab-

you could also use enzymes that do not cut mC's, like HhaI.

Shearing works well too. HydroShear or 21 gauge needle.

-sneth-