Tandem integration of a DNA insert - How can I demonstrate that? (Dec/08/2007 )

Hi friends,

I subcloned a 2kb DNA fragment between two different restriction sites and I suspect that it was cloned in tandem. This is because when the insert is released the insert band is too intense. The problem how can I verify that?

If you choose a primer within your insert directed toward the vector backbone and sequence with it, then if there is a tandem repeat, the sequence will become unreadable at the restriction enzyme cloning site.

It means: many times, repeatedly. When you clone an insert you always have the chance of tandem integration of the insert. This is more likely to happen when the ratio of insert to vector is high. When I analized my transformants through releasing the insert with restriction enzymes, I realized that many of my colonies had a too intense insert band. That could mean that my insert was integrated in tandem. But the question is how can I be sure about it?

You mean that you have 2 or more of your insert per backbone instead of one per backbone??
You should be able to check it by cutting with 2 enzymes in the backbone. If the fragment which contains the insert is longer than expected, then that is what you mean I guess. But you said that the insert has 2 different restriction sites. I don't know how a tandem insert would be possible (unless those 2 enzymes have complementary restriction sites, i.e. SalI-XhoI).

Let me explain my theory. Let's say that my insert is cloned between xho and salI. Normally I would get this construct:

_____ Xho Xho--------SacI SacI_____

where ------ is insert DNA, and
____ is vector DNA

As the concentration of the insert is high, eventually it could happen something like this:

_____ Xho Xho--------SacI SacI-------Xho Xho---------SacI SacI________

If you note, it is only possible insertion of odd number of units.
This is only an idea of mine. I could be wrong but it seems to be OK. Besides If it's true maybe I could have an artificial trimer of my protein!!

find a restriction enzyme that cuts your insert once, asymetrically is best. Find a second enzyme that cuts in your only vector at a known location, such that the you produce a fragment of known size.

Draw out a vector with trimer insertion. Make sure the a double digest using both enzymes produces a clear profile that is different from a single insertion event.

Run the digest.

Double insert and triple insertion events do occur (even PCR skipping over parts of the insert, should a repeat just happens to be there). Rare events but they do happen. This is why all newly constructed plasmids, in my mind have to be checked with at least two different digest paterns.

Why don't you cut the clone using restriction enzymes at the multiple cloning sites other than the ones you used to clone and they should also be the ones that are outside the two sites you used.

For example if your Multiple cloning site reads the following enzymes:...........E1..E2...E3.......E4...E5....E6...E7...............

Say for example you cloned between sites E3 and E4.

If you want to find out whether you have tandem inserts cloned then digest the same clone using E2 and E5 / E1 and E6, etc., Any enzyme other than E3 and E4.

Ofcourse these other enzyme sites should not be in your insert.

This is the most straight forward way to find out if you get 1x, 2x or 3x size of your insert.

I think this is the most simple.....or am I missing something???

Happy cloning.
Rami
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