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DNA Cloning

  1. Michael Andrew Quail

Published Online: 23 SEP 2005

DOI: 10.1038/npg.els.0005344

eLS

eLS

How to Cite

Quail, M. A. 2005. DNA Cloning. eLS. .

Author Information

  1. The Wellcome Trust Sanger Institute, Cambridge, UK

Publication History

  1. Published Online: 23 SEP 2005

This is not the most recent version of the article. View current version (15 DEC 2010)

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Figure 1. A schematic representation of a typical cloning experiment. (a) The vector is cut ([DOWNWARDS ARROW]) within its multicloning site (MCS). (b) The target DNA is cut ([DOWNWARDS ARROW]) so as to produce termini compatible with the vector. (c) The vector and insert are ligated to produce recombinant DNA. (d, e) Recombinant DNA is introduced into appropriate host cells. In this illustration the vector encodes resistance to an antibiotic, X. (f) If the cells are plated out onto medium containing X, only cells that have been transformed will grow and divide to form colonies (groups of around a million cells or ‘clones’ that have arisen from the same original transformed cell).

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Figure 2. Schematic diagram depicting (a) blunt end ligation and (b) ligation of cohesive (‘sticky’) ends. (a) Two double-stranded molecules with blunt ends are ligated to produce a single molecule. (b) 5′ protruding sticky ends, which were created by digestion with the enzyme EcoRI, are ligated to produce a single double-stranded DNA molecule with the EcoRI recognition site being recreated.

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Figure 3. Diagram of the plasmid cloning vector, pUC18. This 2.69 kb double-stranded, closed-circular DNA molecule has an origin of replication (ori) allowing multicopy propagation in E. coli, a β-lactamase gene (ampR) giving resistance to the antibiotic ampicillin and a lacI gene, the product of which induces the expression of the lacZ gene, which in turn encodes β-galactosidase. The multicloning site (dark bar, shown expanded to the right) is close to the 5′ end of the lacZ gene, such that insertion at any of the points within the multicloning site (MCS) should disrupt β-galactosidase expression, thereby allowing blue/white recombinant selection on media containing the colorimetric substrate X-gal and inducer isopropyl-β-d-thiogalactopyranoside (IPTG). The MCS contains closely nested restriction sites for a number of enzymes, in the order shown. It is flanked by sequences to which the M13 forward and pUC reverse primers bind, facilitating polymerase chain reaction amplification or sequencing of the inserted DNA.

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Figure 4. Diagrammatic illustration of the bacteriophage lambda cloning vector λZAPII (Stratagene). This 39.9 kb linear DNA molecule can accept inserts up to 10 kb. It has a multicloning site (MCS) (expanded above) flanked by T7 and T3 polymerase promoters to which T7 and T3 primers bind. The MCS is within the lacZ gene allowing blue/white selection of inserts. The region of the vector containing the insert can be excised to the plasmid pBluescript (SK-) as shown, by infection of cells harboring λZAPII with a helper phage. λZAPII also contains terminal cos sites, lambda genes A–J and a temperature-sensitive mutation within the cI repressor that represses lysis.

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Figure 5. Diagrammatic illustration of the bacterial artificial chromosome (BAC) cloning vector pBACe3.6. This 11.49 kb double-stranded, closed-circular DNA molecule has an origin of replication (ori) allowing single-copy propagation in E. coli, a gene encoding resistance to the antibiotic chloramphenicol (cmR) and loxP recombination sites that flank the region into which DNA is inserted. Dual multicloning sites (MCS) flank a pUC stuffer fragment. The parent vector possesses this region to allow multicopy growth in E. coli allowing easy preparation of the vector. Restriction of the vector with any of the MCS restriction enzymes excises this fragment. The MCS is flanked by T7 and T3 polymerase promoters, to which T7 and T3 primers bind, and is located between the sacBII gene and its promoter so that inserted DNA disrupts expression of its cytotoxic product, ensuring that only recombinants grow on media containing sucrose.