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5. Besrkive hvordan kloningsvektorer bruges til at lave rekombinant DNA. Beksrive følgende: restriktionsenzym-sites i vektoren, kloning ved ligering af rektriktionsfragmenter, brugen af DNA-ligase, insertionel inaktivering af indikatorgen (fx. antibiotisk resistensgen eller lacZ-gen)
Devlin, s.288-9, fig. 7.7
Stryer, s.152-3, fig. 153
Devlin, s.294-fig. 7.10

Cloning vector – a carrier DNA which is employed in incorporating a recombinant DNA into a cell that allows replication of a recombinant DNA. A vector can be prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction endonuclease. The essential feature of a vector is that it can replicate autonomously in an appropriate host.

Bacterial plasmids are ideally suited as recombinant DNA vectors. Plasmids, the bacterial accessory chromosomes, are usually composed of only few thousand base pairs and are rarely associated with the large chromosomal molecule in the bacteria. Genes within a plasmid have various functions that include the ability to confer antibiotic resistance to the bacteria. Plasmids replicate independently of the replication of the main bacterial chromosome.

One type of plasmid – relaxed-control plasmid, is used as cloning vector. It is present in tens to hundreds of copies per bacterium and its replication is solely dependant on host enzymes with long half-lives.
 

As already said, the vector can be prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The interest DNA is also presented for the same restriction enzyme.

The staggered cuts made by the enzyme on the vector and the interest DNA produce complementary single-stranded ends (cohesive ends) that have specific affinity for each other. The DNA fragment and the cut plasmid can then be annealed and joined by DNA ligase, which catalyses the formation of phosphodiester bond between the free 3’-OH group of the interest DNA and the 5’-phosphryl group of the vector DNA on both strands.  

Insertional inactivation – when the insertion of foreign DNA fragment into a vector disrupts a functional gene sequence, the resulting recombinant DNA does not express the original vector gene. If the vector has two antibiotic resistance genes, destruction of one of them and retention of the second allows for the detection of bacterial colonies carrying the foreign DNA of interest within the replicating recombinant vector.

I will take the example of pBR332, which is one of the most used plasmids for cloning. It contains two genes for resistance to tetracycline and ampicillin. Different endonucleases can cleave this plasmid at variety of unique sites, at which DNA fragments can be inserted.

If a specific restriction enzyme cleaves a site which is located in the fx. tetracycline coding gene, the gene becomes inactivated. This effect is called insertional inactivation.

Cells containing pBR332 with a DNA insert in the tetracycline gene are resistant to ampicillin but not tetracycline, so they can be readily selected. Cells that failed to take up the vector are sensitive to both antibiotics, whereas cells containing pBR332 without a DNA insert are resistant to both.
 

Another used vector is the lambda phage.

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