|
|
|
|
Integration of the DNA Insert Into the Vector - Once the DNA fragments to be cloned are prepared and the appropriate vector is selected [the selection will be based on the purpose of cloning and the host(s) to be employed], the DNA segments will have to be integrated into the vector at an appropriate site. The vector is cut open with a restriction enzyme that has a unique (single) target site located in the sequence where the DNA insert is to be integrated.
There are five possible situations with respect to the vector and the DNA insert:
(1) both have completely matched or compatible cohesive ends,
|
(2) one end each of the vector and the DNA insert are compatible and protruding, while their other ends are different from their first ends but are protruding and compatible,
(3) they have different, i.e., unmatched cohesive ends,
(4) both have flush or blunt ends, and
(5) one end of both is cohesive and matched, while the other is blunt. The strategy of integration of the DNA insert in these situations is briefly described below.
|
Both Ends Cohesive and Compatible The simplest strategy concerns the presence of compatible cohesive ends both in the vector and the DNA insert. This happens when the vector is cut open by the same restriction enzyme that was used to isolate the DNA insert. The opened up vector and the DNA insert are mixed under annealing conditions which allow pairing between the compatible cohesive ends of the vector and the DNA insert.
The nicks (the broken covalent bond between the 5'-phosphate and the 3'-OH of two neighbouring nucleotides within a DNA strand) remaining after annealing are sealed, joined or ligated by DNA ligase.
Initially, DNA ligase from E. coli was used, but now-a-days T4 (an E. coli phage) ligase in preferred. DNA inserts joined in this manner can be easily and precisely isolated from the recombinant DNA using the same restriction enzyme that was used to generate the inserts and to open the vector.
|
|
In the recombinant DNAs produced in this way, the DNA insert may be present in either of the two orientations relative to the sequences of the vector. If the two cohesive ends of the vector are marked as 1 and 2 and those of the DNA insert as l' and 2', the insert may join the vector to either yield 1-1' and 2-2' junctions or 1':2' and 2-1' junctions.
The orientation of DNA insert within the vector is not important when only copies of the insert are to be obtained. But it is extremely important when expression of the DNA insert is desired.
|
|
|
In addition to the formation of recombinant DNA, the cohesive ends of the vector itself will pair together to produce unaltered vector molecules. Similarly, the two ends of the DNA insert will also join to yield a circular DNA insert molecule.
Circularised insert is not a problem since it lacks an origin of replication and, as a result, is diluted out of the transformed cells.The formation of unchanged circularised vector can be prevented by treating the opened up vector with alkaline phosphatase, which removes the 5'-phosphate present as monoester at the vector ends.
When such a vector is mixed, annealed and ligated with the DNA insert, two nicks remain in the recombinant DNA due to a lack of phosphate at the 5'-ends of the vector. These nicks are readily repaired once the recombinant DNA is introduced into appropriate host cells.
|
Both Ends Cohesive and Separately Matched One protruding end of the vector may be compatible with one end of the DNA insert, while the other end of vector is compatible with the second end of the insert. This situation arises when one end of the vector as well as that of the DNA insert is generated by one restriction enzyme, while their other end is cut by a different enzyme.
As earlier, the opened vector and DNA insert are mixed under annealing conditions to allow pairing between the compatible ends of the vector and the DNA insert; T4 ligase is then used to seal the nicks to yield recombinant DNA.
In such a situation, only the recombinant DNA is circularised (since the two ends of vector are unmatched as are the two ends of DNA insert) and DNA insert is integrated in only one orientation or direction.
Both Ends Cohesive and Unmatched Often the DNA insert is prepared using one restriction enzyme, while the vector is opened with another enzyme; this generates cohesive ends in the vector and the DNA insert, which are unmatched. In this situation, the following approaches are available.
(1) The protruding ends are converted into blunt ends either by removing the protruding ends by digestion or by extending the recessed ends using Klenow fragment or reverse transcriptase; the blunt ends can then be joined together by T4 ligase.
(2) The blunt ends so produced can again be changed into protruding ends by 3'-tailing. A poly-T tail may be added to the 3'-ends of, say, vector, while poly-A tails are added to the DNA insert; the two now have matched protruding ends, which pair together under annealing conditions to yield recombinant DNA. Often the tails are of different sizes; therefore, the gaps remaining in the recombinant DNA are filled with DNA polymerase I prior to ligation. Alternatively
(3), linkers can be attached to the blunt ends of the vector and the DNA insert. The linkers are cleaved with the appropriate restriction enzyme to generate protruding ends that are compatible; the vector and the insert are now joined together. In addition,
(4) appropriate adaptors may be joined to the protruding ends of the vector and/or the DNA insert to generate completely matched cohesive ends.
Both Ends Flush/Blunt Flush or blunt-ended vector and DNA insert can be joined together by T4 DNA ligase. However, high concentrations of both the enzyme, and the vector and insert DNAs are required.
The DNA insert gets ligated in either orientation, and it can be easily and precisely separated from the vector only if the two were cleaved by the same restriction enzyme (producing blunt ends). Alternatively, the blunt ends can be converted into cohesive ends as outlined above.
One End Cohesive and Compatible, the Other End Blunt If the vector and the DNA insert have one compatible and cohesive end, and one flush end, their cohesive ends pair together when they are mixed under annealing conditions. The T4 ligase seals the nick at the cohesive end and joins the blunt ends as well. It should be noted that under such a situation, only the recombinant DNAs are produced since vector and DNA insert molecules can not circularise due to their one cohesive and one blunt ends.
|
|
