Question

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

Suppose the insert (striped) encoded a protein, can it be transcribed even if the nicks are there?
What would happen to the nicks during DNA replication?
Would the nicked plasmid be repaired somehow? Is there a mechanism that can recognize and fix the ddNTP nick?
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.

Answer 1

TA cloning creates overhangs using ddNTPs to prevent additional nucleotide addition. DNA ligase cannot seal nicks with ddNTPs due to the lack of 3' OH but transcription can occur because RNA polymerase does not need to cross nicks. Modern TA cloning still uses ddNTPs to ensure accurate ligation of PCR products with single-base overhangs.

Step-by-step explanation:

TA cloning is a method used to clone DNA fragments that have been amplified by PCR (Polymerase Chain Reaction). In TA cloning, ddNTPs (dideoxynucleotide triphosphates) are employed to create a single T overhang on the vector by preventing the addition of further nucleotides due to the absence of a 3' hydroxyl (OH) group, which is essential for the formation of phosphodiester bonds during DNA synthesis. Nevertheless, when it comes to the cloning process, some nicks in the backbone that incorporate ddNTPs cannot be ligated by DNA ligase, because ligase requires a 3' OH and a 5' phosphate group present on adjacent nucleotides.

Despite the nicks, transcription by RNA polymerase, such as T7 RNA polymerase, can occur if the promoter is intact and upstream of the insert. The enzyme synthesizes RNA by moving along the DNA template without the need to cross the nicks, thus the insert can be transcribed. During DNA replication, most DNA polymerases are capable of replicating across small gaps or nicks, and the replication machinery, especially DNA ligase, can repair nicks during the replication process, ensuring the integrity of the DNA molecule. Nonetheless, not all cases of nick repair are efficient, and sometimes the repair mechanisms can fail, potentially leading to a truncated replication product.

In modern TA cloning protocols, companies like NEB still use the method of adding ddNTPs to create the overhangs on the vector DNA, because it is a simple and effective way to ensure single-base overhangs for ligation with PCR products that have complementary overhangs. When discussing the repair of nicks, cellular processes such as those involving DNA ligase, are presumed to facilitate the ligation of any remaining nicks not involving ddNTPs — provided that there is a free 3' OH and a 5' phosphate.