Gene cloning or recombinant DNA technology is the joining of two or more segments of DNA to generate a single DNA molecule capable of autonomous replication within a given host (1). Ligase enzymes catalyze the joining or ligation of DNA or RNA segments, where phosphodiester bonds are formed between adjacent 3′-OH and 5′-phosphate termini of DNA and RNA. The most widely used of such enzymes is T4 DNA Ligase. This enzyme can join DNA fragments having cohesive or blunt ends. It requires ATP as the energy yielding cofactor and Mg2+ ions for activity.
A good general vector for DNA cloning should contain basic elements, such as: an origin of replication enabling replication in bacteria; a multiple cloning site (MCS) with an array of unique restriction enzyme sites suitable for cloning of a double-stranded DNA insert and; selectable markers such as antibiotic resistance genes (tetr and ampr) (2).
Additional elements of cloning vectors include a phage-derived f1 origin of replication for the production of single-stranded DNA and a MCS containing a DNA segment from the E. coli lacZ operon that encodes the amino-terminal fragment of β-galactosidase. This lacZ fragment can be induced by isopropylthio-β-D-galactosidase (IPTG), and complements a defective form of β-galactosidase encoded by the E. coli host, allowing blue/white colony or plaque selection of clones (2). In addition, some plasmids contain bacteriophage promoters adjacent to the MCS, so that foreign DNA cloned within this site can be transcribed in vitro (2).
Depending on the size of the DNA to be cloned, DNA fragments are inserted into plasmid (~0.1–10kb), lambda phage (~0–23kb), or cosmid (~35–50kb) vectors. Common cloning vectors such as the pGEM® series(d), pBR322, pUC/M13, pSP series(d), pET series(e), and others can easily replicate DNA inserts of 0–10kb. The multiplication rates of large recombinant plasmids slow as the plasmids get larger, with those that have lost large pieces of their foreign DNA eventually predominating. Larger DNA inserts, up to 15kb, are more easily cloned in lambda vectors, without affecting the normal packaging of the lambda chromosome into functional virus particles (4). Still larger DNA inserts, up to 50kb, can be cloned into cosmid vectors, constructed from the two ends of the lambda chromosome (3). Very large DNA fragments can be cloned into P1, BAC and YAC vectors.
The insert sequences can derive from practically any organism. They may be isolated directly from the genome, from mRNA by reverse transcription, from previously cloned DNA segments (subcloning) or from synthetic DNA sequences (1).
The following discussion focuses on DNA cloning in E. coli hosts, using common plasmid vectors and T4 DNA Ligase. For a discussion on cloning in other bacterial hosts, yeast, plants, Drosophila, and viruses, please refer to reference 4. Figure 2 provides essential information on the cloning of DNA fragments into plasmid or phage vectors, including basic directions on 1) digestion of vector and insert DNA; 2) conversion of 5′- or 3′-overhangs to blunt ends; 3) vector dephosphorylation and; 4) ligation of vector and insert DNA.
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