DNA Cloning

Abstract

Deoxyribonucleic acid (DNA) cloning is the art of creating recombinant DNA molecules that can be introduced into living cells, replicated and stably inherited, such that multiple ‘clonal’ copies of that DNA are produced. Here, DNA of interest is spliced into a DNA vector that has the ability to replicate in host cells and be inherited into future generations of those cells.

This article describes the theory behind cloning; the preparation of vector and insert DNA for cloning; the techniques that can be used to create recombinant DNA molecules (including traditional ligation of blunt and sticky ends, TA cloning, topo cloning, ligation‐independent cloning, recombineering and gateway cloning); introduction of recombinant DNA molecules into host cells; the major classes of vectors available and the variety and utility of the various features and that can be present in those vectors.

Key Concepts:

  • DNA can be joined, or ligated, together to form a recombinant DNA molecule.

  • When cloning DNA, that DNA is ligated to a vector molecule allowing it to be subsequently immortalised.

  • Recombinant vector:DNA constructs can be introduced into living cells, so‐called host cells, through the process of transformation.

  • Once introduced into host cells, the DNA and vector construct is immortalised and can be replicated and stored.

  • Different vectors allow for introduction and maintenance in different host cells.

  • A range of vectors are available, or can be constructed, that contain a variety of elements that confer upon that clone its usefulness. Such elements may include one or more of the following: sequences that control replication and inheritance within the host cell and sequences that allow selection of the recombinant molecule, for example, antibiotic resistance genes, reporter genes and promoter sequences that drive expression of a gene that has been cloned.

Keywords: recombinant DNA; vector; phage; library construction; artificial chromosomes

Figure 1.

A schematic representation of a typical cloning experiment. (a) The vector is cut (↓) within its multicloning site (MCS). (b) The target DNA is cut (↓) so as to produce termini compatible with the vector. (c) The vector and insert are ligated to produce recombinant DNA. (d) and (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).

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.

Figure 3.

Schematic illustration of topo cloning. Vectors that have topoisomerase I sites at their termini are precharged with topoisomerase such that when a compatible DNA insert is introduced the vector and insert are quickly covalently attached, ejecting the topoisomerase. Various vectors are available from Life Technologies that allow (a) blunt, (b) directional and (c) T/A cloning. Reproduced with permission from Life Technologies.

Figure 4.

Schematic diagram depicting the technique of ligase‐independent cloning (LIC) system as marketed by Merck. The target vector has a specific 5′ overhang sequence that lacks adenosine bases and complementary inserts can be generated following PCR with specific 5′‐primer sequences and end‐repair with T4 DNA polymerase and dATP. The 3′–5′ exonuclease activity of the polymerase specifically removes those bases that are not supplied and halts when it reaches the first adenosine base as these nucleotides are present in the reaction mixture. Cloning is very efficient because only the desired product is formed by annealing. The annealed LIC vector and insert are transformed into competent E. coli cells. Covalent bond formation at the vector:insert junction occurs within the cell to yield circular plasmid. Reproduced with permission from EMD Chemicals Ltd.

Figure 5.

Diagrams describing the Gateway cloning system. (a) The system is based on the integrase enzyme system of phage lambda that recognises specific attachment (or att) sites and catalyse reversible integration of the phage genome into its E. coli host. (b) Sequences of interest are first introduced into an entry clone. This can be done by BP cloning (after attB sequences are added to the termini of your sequences by PCR), topo cloning, traditional cloning or by buying premade libraries. (c) LR clonase then catalyses the simple and specific transfer of sequences flanked by entry clone attL sites into destination vectors that contain attR sites. (d) A range of destination vectors are available that support a range of different investigations. Reproduced with permission from Life Technologies.

Figure 6.

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.

Figure 7.

Diagrammatic illustration of the bacteriophage lambda cloning vector λ ZAPII (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 harbouring λ ZAPII with a helper phage. λ ZAPII also contains terminal cos sites, lambda genes A–J and a temperature‐sensitive mutation within the cI repressor that represses lysis.

Figure 8.

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.

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Further Reading

Birren B, Green ED, Klapholz S, Myers RM and Roskams J (eds) (1997) Genome Analysis: Analysing DNA, A Laboratory Manual, vol. 1. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Brown TA (2000) Essential Molecular Biology: A Practical Approach. Oxford, UK: Oxford University Press.

Markie D (ed.) (1996) YAC Protocols. Methods in Molecular Biology, vol. 54. Totowa, NJ: Humana Press Inc.

Web Links

ATCC (American Type Culture Collection). A global bioresource center. http://www.atcc.org.

Invitrogen gateway cloning introduction. http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Cloning/Gateway‐Cloning/Gateway‐Technology.html

Jackson Lab introduction to cre‐lox recombination. http://cre.jax.org/introduction.html

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How to Cite close
Quail, Michael Andrew(Dec 2010) DNA Cloning. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005344.pub2]