DH5 alpha methylation profile - (Jan/31/2008 )
I'm not a molecular biology specialist, so I apologize if my question is a bit stupid, or have ever been asked on this forum.
I work with a pNL4-3 vector (with complete HIV genome inside), which is produced by a E Coli DH5 alpha strain. I have extracted the plasmid DNA from bacterial cultures and at the moment, I'm trying to digest it by ApaI and SalI. Logically, we should observe two equal bands (about 3700 and 11000 bp long). After the digestion, there is 1 large band at about 4000 bp, and a very small band (sometimes nothing) between 16000 and 23000bp (I use DNA Marker "lambda Hind III").
And a single digestion with ApaI never gives us a single band 15000 bp long...
Does the problem comme from the digestion (I have already tried to increase the enzyme quantity and the time of digestion, but with no result) ?... or from DNA methylation by bacterial cells? In this case, what can I do (I must work with these 2 restriction enzymes)? Do you know any "non-methylater" bacterial strains that could be used?
Thank you for your answers, and good luck in your experiments
ApaI can indeed be blocked by methylation. If the recognition site in the plasmid is followed by (A/T)GG the site will be methylated and ApaI will not cut there. So if that's the case in your situation, you should transfer the plasmid into a dcm- strain, for which we normally use the INV110 strain from Invitrogen
pNL4.3 is a commonly used vector for HIV-1 work, but the problem is that a lot of different clones exist. I faced the same problem as you did some years ago, just to find out that the sequence we had isn't correct, within the HIV-1 part there were some mutations and also there was a lot of extra genomic DNA between the LTR's and the pUC-backbone (so I got bigger bands than expected). So, if possible, try to sequence your plasmid (or do some basic PCR's on just to check), because you might be stuck with a clone that IS 20kb+ but still has the correct NL4.3-genome.
Thank you for your answers!
I forgot to mention that I use pNL4-3 including NY5-LAV strain: ApaI restriction site GGGCCC is immediately followed by CT AGGAAAAA. Must the methylation site A/TGG be just after the site to impair enzyme action?
I read in a website that ApaI cleavage activity was "impaired" and not "blocked" by this type of methylation: do you know a protocol to increased enzyme activity (increase enzyme concentration...)
It looks like for this strand, the site will not be methylated but you should also check the reverse strand, as the methylation recognition sequence (CC(A/T)GG) can also be formed at the other side (so in front of the ApaI site but on the other strand). According to what I know, methylation will completely block ApaI cleavage (as I read in the NEB catalog) but I may be wrong about this.
I forgot to mention that I use pNL4-3 including NY5-LAV strain
Still, there can be big variation in the entire sequence! Sad but true (I've struggled quite some time with this issue, using ApaI too actually). So, before proceeding I suggest you try to find out, using other restriction enzymes (eg: EcoRI cuts halfway and XhoI cuts in nef for example, both should cut only once and are not affected by methylation) how big your molecular clone is.
You're right, Vairus...a colleague had the same idea this morning, and I test three different enzymatic digestions this morning. I'm waiting for the results...
Digestion experiments of pNL4-3 give me strange results: I need (4µL x 80U/µL)= at least 320U of EcoRI to open this plasmid...I precise that I used for digestion 1µL of pNL4-3 (1µg/µL, I'm sure of this). According to the manufacturer instructions, 0.1-1µL (with enzyme concentration= only 10U/µL) sould be sufficient. And EcoRI is not methylation sensitive.
What a strange plasmid ! Do you have any explanation or suggestion?
thank you for your answers
Something in your prep is inhibiting EcoRI then, or your enzyme has been stored improperly, or maybe (due to the presence of 2 LTR's) some re-organisation of the plasmid has taken place so that you're seeing strange results (E. coli does strange things with long terminal repeat sequences, maybe you should grow in different cells? I have no problems with pNL4.3 growing in DH5-alpha at 30°C and shaking at 180 rpm, but growing at 37°C and shaking 225-300 rpm can cause problems for plasmids with repeats).