What are common causes of unexpected ligation products?

Question

I digested two plasmids, one with EcoRI and AgeI and the other with EcoRI and XmaI. Digests looked as expected, so I purified the respective fragments and set up the T4 DNA ligation (AgeI and XmaI sites are compatible).

After E.coli transformation, culture and miniprep from 12 of the resulting cultures, I digested the isolate from the minipreps with BclI to confirm I had the correct product (because there was one BclI site in the insert and another one in 1.4kb away in the backbone). The results of this digest clearly show that there are two different ligation products: four lanes share one banding pattern and the other eight share a pattern which is different from it.

I checked both plasmids for other restriction sites of the enzymes used which I overlooked before, but found none. Because EcoRI and XmaI/AgeI sites are radically different (AATT vs. CCGG), I would exclude the possibility of the insert being ligated in reverse orientation.

Are there any common causes of unexpected ligation variants besides unnoticed restriction sites and reverse ligation?

Answer 1

Could be insert polymerization. If you have your stretch of DNA like this:

 (5')-AATTagctagcatcgtgatcgacg-(3')
          ||||||||||||||||||||
     (3')-tcgatcgtagcactagcagcGGCC-(5')

And you take that, flip it around, it will ligate onto itself, like this:

 (5')-AATTagctagcatcgtgatcgacg-(3')(5')-CCGGcgacgatcacgatgctagct-(3')
          ||||||||||||||||||||              ||||||||||||||||||||
     (3')-tcgatcgtagcactagcagcGGCC-(5')(3')-gctgctagtgctacgatcgaTTAA-(5')

This is easy enough to check for. Just calculate the size fragments you'd expect from a vector with two inserts (there'd be an extra BcII cutsite now, and the extra fragment would probably be the size of the insert). If this is just out of curiosity, and one of your things is the correct size, I'd ignore the ones that didn't work and go ahead with your ones that did.

If you do have a problem with it, or you are getting all your clones polymerizing, you can treat your insert with an alkaline phosphatase. This will eat off the (5') phosphate that the ligase needs to stick the on to the (3') hydroxyl. The result is that an insert cant bind to another insert, but once it finds a vector it will be able to ligate:

 insert to insert:    AATTagctagcatcgtgatcgacg-(3') :( CCGGcgacgatcacgatgctagct-(3')
                         ||||||||||||||||||||              |||||||||||||||||||| 
                    (3')-tcgatcgtagcactagcagcGGCC  :( (3')-gctgctagtgctacgatcgaTTAA

 insert to vector:    ....ctagct-(3')     AATTagctagcatcgtgatcgacg-(3')(5')-CCGGcgacg....
                      ||||||||||              ||||||||||||||||||||              |||||||||
                      ....gatcgaTTAA-(5')(3')-tcgatcgtagcactagcagcGGCC     (3')-gctgc....

There will be nicks on the top left, and bottom right, but the bug you put it into should clean those up for you. You can get alkaline phosphatase for fairly cheap from lots of places, NEB is always a safe bet.

Answer 2

A second investigation has suggested that the ligation product in question was indeed the vector, without ligated insert!

The vector also contained a BamHI site close to the XmaI site, so in a subsequent digest with EcoRI and BamHI, I was expecting to see the same band sizes that I ligated together in the first place.

All colonies contained plasmids that produced the long (vector) band, and the same eight as before produced the shorter (insert) band as well. However, those four which were aberrant in the previous experiment were this time lacking the shorter band.

This means the eight colonies with two bands contained the desired ligation product, while the other four contained a plasmid which was restricted at a single site (undigested it would have produced three bands like the control).

As the BamHI site was in the vector and untouched throughout the whole process, I conclude that those four were missing the EcoRI site, which should have been regenerated during the ligation, and hence indeed the EcoRI site and the XmaI site must have been ligated without insert somehow.

Long story short

This means that either

• An exonuclease digested the sticky ends of the vector away, or
• The sticky ends simply degraded by chance

resulting in a blunt-ended re-ligation of the vector without insert and consequently loss of the EcoRI site.

I believe these to be quite important things to consider in cloning - in subsequent experiments I have had several colonies growing on negative control plates (i.e. colonies transformed with a control ligation that contained no insert), all of which were probably due to loss of sticky ends and blunt ligations, resulting in functional plasmids that conferred ampicillin resistance despite containing an undesired plasmid.