Insert only ligating in backward direction - (Sep/10/2007 )

Hello all,

Another person in our lab was working on cloning a gene into a vector, however, she has since left, leaving me to finish the project. It's oh so close to being finished, but I have a major hang-up.

Here was her process:

1.) Designed primers with NotI sites at each end of the gene and amplified.
2.) Excised existing gene from vector (also flanked by NotI on each end) and discarded.
3.) Treated vector with SAP.
4.) Cut PCR product with NotI and ligated.

Despite treating the vector with SAP, she had mostly self-ligation of the vector (later she discovered she used too much DNA, so the enzyme reaction was not efficient). She did find one clone that contained the insert using colony PCR. She had it sequenced and everything was perfect, but the insert was backwards. Rather than redo the whole PCR process, I suggested she take this plasmid and excise the insert with NotI, gel purify, and religate, hopefully getting some clones with the insert the right direction. She did this, used less vector in her SAP treatment, and got 6 colonies, and no colonies on a vector only control plate. She screened all the colonies using a directionality screen (using the upstream amplification primer of the gene and a sequencing primer that anneals to the vector downstream of the insertion site. According to her, this should only produce a band if the insert is in the right direction (I don't know much about this technique). All 6 colonies came up positive, but she couldn't proceed further as she left our lab.

So here's where I come in: I miniprepped all 6 clones, and did a restriction digest with EcoRI to assess directionality my way. The vector has an Eco RI site and the insert has one just downstream from the start codon, so directionality is pretty easy to assess based on the sizes of the bands produced. To my dismay, all 6 produced bands consistent with the insert being backwards! Aargh! I don't know why her screen was positive, but I trust my method more. I could have them all sequenced, but I think that would be a waste of time.

So my question is, why are they all backwards? Are there circumstances in which an insert with the same RE site on both ends can preferentially ligate in one direction vs. the other? It's possible it's just bad luck, I guess you could flip a coin 6 times and have it come up heads each time, but the odds of that are still pretty low.

Anybody have any ideas? I hope I've made some sense.

Cheers

fishboy83

Two things can be happening,
1 - Bad luck
Build enough plasmids and you will sometimes see such a run. It could be bad luck. Do you have more colonies that you can look at?

2. DNA Tertiary structure
Again, this phenomena is not unknown. Others aside from yourself have reported such occurance in the forum. Due to the comformation of DNA fragment, it perferentially ligates in one orientation. I personally, have one fragment such as this, though thankfully the perferred orientation is the desired orientation. However all hope is not lost, while one orientation is perferred, you can still find the other orientation if enough clones are looked at.

What to do?
The cheapest way would be to look at more colonies. If you don't have anymore colonies, do the ligation again (with the SAP step corrected). If it is bad luck, the string of 6 in the wrong orientation will not happen again. If it is tertiary structure you will have to look at a lot more clones. Try 24 colonies.

If you can not find clones in the correct orientation, you will then have to consider making the insert directional by buying a new primer.. one adding a different restriction site. Can the old gene in the vector be remove by 2 restriction enzymes?

It is also possible that the "correct" orientation produces a toxic gene product, killing the cells containing that plasmid. Since you don't get a colony, it looks like bad luck, but you really had ok luck which was selected against.