What Is a Recombinant Plasmid?

There is a gene of interest, a plasmid, and then the two are connected by an enzyme is a recombinant plasmid. Recombinant plasmid is a kind of action, method, and a noun.

Recombinant plasmid

Fragments with non-complementary overhangs

There are several strategies for the ligation reaction of foreign DNA fragments and plasmid vectors:

1. Fragments with non-complementary overhangs Digestion with two different restriction enzymes can produce non-complementary sticky ends. This is also the most easily cloned DNA fragment. In general, commonly used plasmid vectors contain Multiple cloning sites consisting of multiple recognition sequences for different restriction enzymes, it is almost always possible to find a vector with a restriction site that matches the end of a foreign DNA fragment, thereby cloning the foreign fragment onto the vector in a targeted manner. During PCR amplification, different restriction sites can be artificially added to both ends of the DNA fragment so as to be connected to the vector.

With the same sticky ends

2. Such ends can be obtained by treating the same sticky ends with the same enzymes or homologous enzymes. Because the plasmid vector must also be digested with the same enzyme and the same two sticky ends are obtained, both the foreign fragment and the plasmid vector DNA may undergo self-cyclization or several molecules in series to form an oligomer during the ligation reaction, Both connection directions are possible. Therefore, the concentration of the two DNAs in the ligation reaction must be carefully adjusted so that the number of correct ligation products reaches the highest level. The 5 'phosphate group of the vector DNA can also be removed by alkaline phosphatase to minimize the self-cyclization of the plasmid DNA. An exogenous DNA fragment with a 5 'end phosphate can be efficiently linked to a dephosphorylated vector, resulting in an open-loop molecule with two gaps, which can be amplified after transfer to E. coli The gap can be repaired automatically during the process.

With flat end

3. The blunt ends are produced by digestion with restriction enzymes or exonucleases that produce blunt ends, or caused by DNA polymerase supplementation. Because blunt end ligation efficiency is much lower than sticky end, the concentration of T4 DNA ligase and the concentration of foreign DNA and carrier DNA are much higher in the ligation reaction. It is usually necessary to add a low concentration of polyethylene glycol (PEG 8000) to promote the aggregation of DNA molecules into aggregates to improve the conversion efficiency.

in special cases

The end of the exogenous DNA molecule and the end of the vector used cannot match each other.You can connect the end of the linear plasmid vector or the end of the exogenous DNA fragment with a suitable linker or adapter to make it match, or you can control it. The large klenow fragment of E. coli DNA polymerase I was used to fill in the 3 'concave end, so that the unmatched ends were converted into complementary ends or blunt ends before ligation.