RE digest and subsequent ligation - A base is now missing from restriction site - how, why? (Jul/14/2006 )

Hi,

I have digested the pQE30 vector (Qiagen) and my insert with both BamHI and PstI. In frame ligation was required so that a hexahistidine tag is added to my gene for downstream protein purification purposes.

I PCR'ed my transformed cells and they looked great (vector and gene specific combination), but following culture they failed to express my protein

So, I got my vector sequenced and it shows that one of the bases in the BamHI restriction site has been lost, which means my gene is now out of frame. I have never heard of this happening before and wondered if anyone else had come across it, or if anyone knows what could have caused it?

THe sequence should have been 5`...GGATCCatg--->my gene
but the sequencing showed 5`...GGATCatg--> my gene.

I find this particularly strange because the BamHI site is the one that i used to ligate my gene into the vector, so I didn't think that the ligation would have been successful if something was wrong with the restriction site!

Is BamHI highly specific for its restrition site? Do you think that the base pair was originally there following ligation but has somehow been lost by mutation / something else? Do you think that this will happen again if i try to do the same digest / ligation again?

Any assistance would be greatly appreciated as I am highly confused

Thanks

This is quite strange.

I have been told that overdigestion with any enzyme could chew up the sites but then one wouldnt find any clone as the ends r no longer compatible for ligation.

I am guessing something happened in the bacteria. Try setting a new ligation with insert and vector digetsed again. Hopefully it should work.

Try asking technical support from the company which supplied the enzymes.

Good Luck !!!

Thanks

Can anyone refer me to any papers which would describe this? I have been looking them up but if i look up mutation, restriction enzyme, I come up with loads of papers about purposefully introducing restriction sites into DNA. WHen I've been looking up overdigestion, I just find info from company websites about how they quality test enzymes, or papers where people are describing using a lot of enzyme.

Can anyone help me? As I am an Honours student and have wasted a lot of time on this (and my supervisors think it is an interesting and unusual result) they told me that I should put it into my thesis at the end of the year. But I don't really have a good explanation at the moment, or any papers to back it up.

Thanks

i dont know of any paper abt overdigestion.

The technical reference for the enzyme would suggest how many fold overdigestion is fine. If its 10x , then u better not overdigest for a very long time.

NEB's technical reference is a good site for information on enzymes.

Hi there,

First of all, if it is overdigestion problem try to digest for 2 hurs. one hour works fine with me.
regarding your question, in the past I tried to clone some inserts into TA vector and what I got was totally strange I got some extra bases in the restriction sites as well as between the restriction sites and the inserts I was trying to clone. I explained that by amplification error due to using Taq polymerease, because after using expand long polymerease I got my inserts with the right restriction sites and subsequently I got my clones.

So, if you are using PCR to introduce your restriction sites try to use high fedility enzyme (Pfu, Pfu ultra or HF from invitrogen)
digest for 2 -4 hours and ligate overnight


I hope that will help

I can't offer an explanation, but I can offer some sympathy...I know how this problem feels!

I digested vector and PCR product with SmaI for ligation...ligated, did transformation, sequenced the construct of one...and stupidly did not notice that one base from my SmaI site at the ATG end of my gene was missing...my perfectly inserted gene was out of frame, and when overexpressed obviously produced a protein that was meaningless and terminated prematurely (which is how I worked out there was a problem).

Grrrr. Stupid me.

So, I sequenced some contructs from other transformant colonies. There were some with one base chopped off my SmaI site, some with 2 chopped off and one, luckily, was perfect....which I used. Live and learn, be super careful when you digest, and always check your sequencing results very, very thoroughly!

Could it be a sequencing error? What happens if you cut your clone with BamHI? If it is a true mutation, the plasmid won't cut, if it is a sequencing error, it will.

I've never heard of "overdigestion" -- I've on occasion digested plasmids overnight with no ill effects.

Did you use CIP at all?

Losing either of the terminal G's on the lower strand would introduce this mutation, which would still leave three (or four) complementary bases for ligation: