Study of Cloning Vectors

Cloning vector is a DNA molecule in which when a foreign DNA is integrated achicves the capability to replicate it within itself to produce many clones of recombinant DNA.

Characteristics of a Cloning Vector

  1. Its size should be small.
  2. It should bear the capability to self-replicate within the host cell.
  3. It should carry a restriction site for the action of restriction endonuclease.
  4. Its replication property should not be compromised by the insertion of donor DNA fragment.
  5. To ease the identification of recombinant cell it should possess some marker gene.
  6. It should bear multiple cloning sites.


Plasmids are naturally derived, extra chromosomal, double-stranded DNA molecules that replicate autonomously in bacterial cells. Their size ranges across a few thousand base pairs to more than 100 kilo-bases (kb).

Plasmid DNA forms replica prior to each cell division just as the host-cell chromosomal DNA does. One copy of the plasmid DNA is divided into individual daughter cell during the cell division. This daughter cell propagates the plasmid through successive generations of the host cell. Plasmids mostly are circular; however, Streptomyces and Borrelia burgdorferi bacteria provide linear plasmids.

The naturally occurring plasmids bear genes that aid the host cells. For example, certain bacterial plasmids encode enzymes responsible for inactivating antibiotics; hence, a bacterial cell with such plasmid develops resistance to the antibiotic and is capable of replicating in antibiotic-rich environment; and if the same bacterium lacks the drug-resistant plasmid, it cannot survive. This property of plasmid favours them to act as cloning vectors.

Properties required in plasmids to serve as cloning vectors are:

  1. Their isolation from the host cells should be easy.
  2. They should possess a restriction site for one or more restriction enzyme(s).
  3. Their replication properties should not be changed upon insertion of a linear foreign DNA at one end of the restriction sites.
  4. They can be reintroduced into the host bacterial cells. Cells bearing plasmid with or without the insert should be identifiable.

Given below are some of the examples of plasmids:

pBR Vector Plasmids: The name pBR was derived from the name of its discoverer, i.e., Bolivar and Rodriguez. Modification of the former plasmids of E. coli (i.e., pBR318 and pBR320) produced pBR322 plasmid of 4362 bp length. Its origin of replication (ori) is derived from ColEI (a naturally occurring plasmid).

The pBR322 plasmid contains genes conferring resistance to antibiotics, like ampicillin (ampr) and tetracycline (tetr). More than 10 enzymes are present with distinctive cleavage sites on the pBR322 genome. The tetracycline resistant gene (tetr) carry the target sites of these enzymes and the promoter of that gene carry sites for further two (ClaI and HindIII).

In the ampr gene, six specific restriction sites are present. With the help of these enzymes cloning in pBR322 causes insertional inactivation of either the ampr or the tetr markers. The host cells with recombinant plasmid grow in the medium either containing ampicillin or tetracycline but not both. Easy selection of recombinants is not favoured by cloning in other unique sites, since none of the antibiotic resistance determinants are inactivated.

The 3´ tetra-nucleotide extensions formed upon digestion are ideal for terminal transferase; therefore the PstI site in the ampr gene in pBR322 plasmid is important for cloning by the homopolymer tailing method. When cloning occurs at HindIII site of pBR322 plasmid, tetracycline resistance is lost.

In certain recombinants however, tetris retained or increased as the HindIII site is present in the promoter rather than the coding sequence. Thus, the occurrence of insertional inactivation depends on whether or not the cloned DNA carries a promoter-like sequence that may initiate transcription of the tetr gene.

pBR322 Vector Plasmid

pUC Vector Plasmids: The name pUC stands for Plasmid University of California as these genetically modified plasmids were first developed in the University of California. Advantages of these plasmids as a vector for cloning are:

  1. They are smaller in size but still can hold relatively large DNA inserts.
  2. In a host cell, pUC (like pUC18) can replicate to form 500 copies per cell, producing numerous clones or copies of inserted DNA fragments.
  3. They bear an ampicillin resistance gene, and an origin of replication (derived from E. coil plasmid pBR322).
  4. They bear a lac Z gene obtained from E. coil. In the lac region, many restriction enzyme sites, known as multiple cloning poly-linker sites have been engineered.
  5. They have a selection system to differentiate between recombinant (plasmid with inserted DNA fragment) and non-recombinant plasmids.
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Santhakumar Raja

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