What are you going to learn?

  • What are cloning vectors?
  • Why are cloning vectors so useful?
  • How do cloning vectors work?
  • Which cloning vectors can we use?
  • terms: selectable marker, bacterial plasmids, transformation, cloning DNA, phage vectors, yeast artificial chromosome (YAC), expression vectors

Cloning vectors are DNA molecules with inserted DNA fragments. They are useful for copying fragments of DNA as they can be introduced into a host cell and replicate. Cloning vectors have 3 important characteristics: a) they contain a restriction site(s) that allows DNA fragments to be inserted, b) they have an origin of replication, which allows them to replicate within the host cell, c) they can carry a selectable marker (for example, an antibiotic resistance gene) that allows them to be distinguished from host cells without the vector.

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cloning vector = a DNA molecule with inserted DNA fragments that can be copied in a host cell
3 important characteristics of a cloning vector

The most important and also the first vectors developed are genetically modified bacterial plasmids. Plasmids are circular, double-stranded DNA molecules that are part of bacteria. They can replicate independently from the chromosomes within bacterial cells they are inserted into. Plasmids are introduced into bacteria by the process of transformation, about which we talk in a different article.

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bacterial plasmids = first vectors developed; inserted into bacterial cells by transformation
a bacterium and its organelles

Cloning DNA with a plasmid begins by cutting both the plasmid DNA and the DNA to be cloned with the same restriction enzyme. DNA fragments are then added to the cloning vector together with DNA ligase. Sticky ends of DNA fragments anneal, and DNA ligase creates phosphodiester bonds to seal the nicks in the DNA backbone. The bacterial plasmid is then introduced into bacterial host cells by transformation. Once inside the cell, plasmids replicate quickly and produce multiple copies of the DNA fragment.

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Cloning DNA with a plasmid:
1) the plasmid DNA and the DNA to be cloned are cut by the same restriction enzyme
2) DNA fragments are inserted into the cloning vector in the presence of DNA ligase
3) DNA fragments anneal
4) DNA ligase seals the nicks in the DNA backbone
5) the plasmid is introduced into host cells by transformation
6) the plasmid replicates and produced copies of the DNA fragment
cloning DNA with bacterial plasmids

Although plasmids are often used in recombinant DNA technology, they are so small that they can only accept DNA fragments up to about 25 kilobases (kb) in size. Therefore, other vectors that can accept larger DNA fragments are used. One of these are phage vectors, for example, bacteriophage λ (lambda) - a double-stranded DNA virus, which infects E. coli, and which can carry DNA fragments up to 45 kb long. Once the fragments are inserted into the phage vector, the phage can infect bacterial host cells, replicate and copy the DNA fragment inserted.

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Phage vectors can carry larger DNA fragments than bacterial plasmids.
cloning DNA with bacteriophage λ

There are also cloning vectors that can be introduced into eukaryotic cells. A yeast artificial chromosome (YAC), a DNA molecule with a yeast origin of replication, a pair of telomers, and a centromere, can carry long DNA fragments and behaves in the same way as yeast chromosomes. Expression vectors are used to ensure that the mRNA of the cloned gene (i.e., of the inserted DNA fragment) is expressed and the proteins it encodes are produced. Both plasmids and phage vectors usually only carry DNA fragments and do not force the cell to transcribe them into mRNA. To be able to initiate both transcription and translation of the cloned gene, expression vectors contain sequences required for transcription and translation in host cells.

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Yeast artifical chromosome (YAC) can be introduced into eukaryotic cells.
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Expresion vectors ensure the mRNA of the cloned gene is expressed.

References:
Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M. A., & Killian, D. (2019). Concepts of Genetics. Pearson.
Pierce, B. A. (2019). Genetics: A Conceptual Approach (Seventh ed.). W. H. Freeman.