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In an undergraduate lab class on TA cloning, it was explained that ddNTPs are used as the substrate for terminal transferase when making the T overhangs of the vector. I was told this was to ensure that the overhang is only 1 bp long.

However, they also say that after the insert binds to the vector, the remaining 4 nicks are ligated. I was immediately confused, because due to the missing 3' hydroxyl groups (consequence of using ddNTP), a phosphodiester bond could not form in 2/4 of the nicks. I may be mistaken, but the oxygen from the 3' hydroxyl is integrated into the bond, so the nick cannot be ligated if it is just a 3' hydrogen. Some people said that ddNTP cannot be polymerised on, but can be ligated.

T.A. Holton and M.W.Graham, 1990 partially answered my question. They confirmed that 2/4 of the nicks cannot be ligated if ddNTP is used (see image).

I have several questions about this

  1. Suppose the insert (striped) encoded a protein, can it be transcribed even if the nicks are there?
  2. What would happen to the nicks during DNA replication?
  3. Would the nicked plasmid be repaired somehow? Is there a mechanism that can recognize and fix the ddNTP nick?
  4. This is a paper from 1990, does modern TA cloning (say, NEB T4 TA cloning protocl) still use ddNTP? If not, how do they assure that the overhang is only 1 bp long?

As for (1), I think perhaps in theory it can be transcribed, as the promoter side of the insert is not nicked, and RNA pol does not need to traverse a nick to access the insert. I suppose the polymerase would fall off the strand at the end? As for (2), I would think that the whole plasmid would fall apart as the replication bubble spreads, unless the nick is repaired somehow prior.

Thanks in advance.

enter image description here

Image taken from figure 1 of T.A.Holton and M.W.Graham, 1990.

T.A.Holton and M.W.Graham, 1990, A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors, Nucleic Acids Research, Vol. 19, No. 15

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To answer question (4):

A-tailing can be done in multiple ways, including the way you described. See here for the most common protocols: https://international.neb.com/applications/cloning-and-synthetic-biology/dna-end-modification/a-tailing

The way I usually do it is to incubate the insert (e.g. from PCR) with Taq-Polymerase, which will always create a single A-overhang. Simply incubate at 72°C for 30 minutes and you are done (no need for ddNTPs, dNTPs are sufficient). Then you can use pre-existing vectors with T-overhangs for a straightforward ligation.

For the other questions I'm not very sure, although when you perform the ligation and transform bacteria with it, there is probably some way they will repair it. The endproduct (after miniprep) will certainly be an intact plasmid.

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