other enzymes may be used to prepare DNA for cloning. In addition to restriction endonucleases, nucleases that degrade single-stranded extensions, such as S1 nuclease and mung bean nuclease, are used to generate blunt ends for cloning (Fig. 2.3A). This is useful when the recognition sequences for restriction enzymes that produce complementary sticky ends are not available on both the vector and target DNA molecules. Blunt ends can also be produced by extending 3′ recessed ends using a DNA polymerase such as Klenow polymerase derived from E. coli DNA polymerase I (Fig. 2.3B). Phosphatases such as calf intestinal alkaline phosphatase cleave the 5′ phosphate groups from restriction enzyme-digested DNA (Fig. 2.3C). A 5′ phosphate group is required for formation of a phosphodiester bond between nucleotides, and therefore, its removal prevents recircularization (self-ligation) of vector DNA. On the other hand, kinases add phosphate groups to the ends of DNA molecules. Among other activities, T4 polynucleotide kinase catalyzes the transfer of the terminal (γ) phosphate from a nucleoside triphosphate to the 5′ hydroxyl group of a polynucleotide (Fig. 2.3D). This enzyme is employed to prepare chemically synthesized DNA for cloning, as such DNA molecules are often missing a 5′ phosphate group required for ligation to vector DNA.
Figure 2.3 Some other enzymes used to prepare DNA for cloning. (A) Mung bean nuclease degrades single-stranded 5′ and 3′ extensions to generate blunt ends; (B) Klenow polymerase extends 3′ recessed ends to generate blunt ends; (C) calf alkaline phosphatase removes the 5′ phosphate group from the ends of linear DNA molecules; (D) T4 polynucleotide kinase catalyzes the addition of a 5′ phosphate group to the ends of linear DNA fragments. Dotted lines indicate that only one end of the linear DNA molecule is shown.
Insertion of Target DNA into a Plasmid Vector
When two different DNA molecules are digested with the same restriction endonuclease the same sticky ends are produced in both molecules. After the two molecules are mixed together, new DNA combinations can be formed as a result of complementary base-pairing between the extended regions (Fig. 2.4). The enzyme DNA ligase, usually from the E. coli bacteriophage T4, is used to reform the phosphodiester bond between the 3′ hydroxyl group and the 5′ phosphate group at the ends of DNA strands that are already held together by the hydrogen bonds between the complementary bases of the extensions (Fig. 2.4). DNA ligase also joins blunt ends, although this is generally much less efficient and typically requires a much greater amount of DNA ligase.
Figure 2.4 Ligation of two different DNA fragments after digestion of both with restriction endonuclease BamHI. Complementary nucleotides in the single-stranded extensions form hydrogen bonds. T4 DNA ligase catalyzes the formation of phosphodiester bonds by joining 5′ phosphate and 3′ hydroxyl groups at nicks in the backbone of the double-stranded DNA.
Ligation of restriction enzyme-digested DNA provides a means to stably insert target DNA into a vector for introduction and propagation in a suitable host cell. Many different vectors have been developed to act as carriers for target DNA. Most are derived from natural gene carriers, such as genomes of viruses that infect eukaryotic or prokaryotic cells and integrate into the host genome, or plasmids that are found in bacterial or fungal cells. Others are synthetically constructed artificial chromosomes designed for delivery of large pieces of target DNA (>100 kilobase pairs [kb]) into bacterial, yeast, or mammalian host cells. Many different vectors that carry sequences required for specific functions, for example, for expression of foreign DNA in a host cell, are described throughout this book. Here, vectors based on bacterial plasmids are used to illustrate the basic features of a cloning vector.
Plasmids are small, usually circular, double-stranded DNA molecules that are found naturally in many bacteria. They can range in size from less than 1 kb to more than 500 kb and are maintained as extrachromosomal entities that replicate independently of the bacterial chromosome. While they are not usually essential for bacterial cell survival under laboratory conditions, plasmids often carry genes that are advantageous under particular conditions. For example, they may carry genes that encode resistance to antibiotics or heavy metals, genes for the degradation of unusual organic compounds, or genes required for toxin production. Each plasmid has a sequence that functions as an origin (initiation site) of DNA replication which is required for it to replicate in a host cell. Some plasmids carry information for their own transfer from one cell to another.
The number of copies of a plasmid that are present in a host cell is controlled by factors that regulate plasmid replication and are characteristic of that plasmid. High-copy-number plasmids are present in 10 to more than 100 copies per cell. Low-copy-number plasmids are maintained in 1 to 4 copies per cell. When two or more different plasmids cannot coexist in the same host cell because they use the same mechanism of replication, they are said to belong to the same plasmid incompatibility group. However, plasmids from different incompatibility groups can be maintained together in the same cell. This coexistence is independent of the copy numbers of the individual plasmids. Some microorganisms have been found to contain as many as 8 to 10 different plasmids. In these instances, each plasmid can carry out different functions and have its own unique copy number, and each belongs to a different incompatibility group. Some plasmids can replicate in only one (or very few) host species because they require very specific proteins for their replication as determined by their origin of replication (often denoted as oriV or origin of vegetative replication to distinguish it from the oriC or origin of chromosomal replication). These are generally referred to as narrow-host-range plasmids. On the other hand, broad-host-range plasmids have less specific origins of replication and can replicate in a number of different bacterial species. The copy number, incompatibility group, and host range of a plasmid are considered when choosing a suitable vector for a molecular cloning experiment.
As autonomous, self-replicating genetic elements, plasmids are useful vectors for carrying cloned DNA. However, naturally occurring plasmids often lack several important features that are required for a good cloning vector. These include a choice of unique (single) restriction endonuclease recognition sites into which the target DNA can be inserted and one or more selectable genetic markers for identifying recipient cells that carry the cloning vector–insert DNA construct. Most of the plasmids that are currently used as cloning vectors have been genetically modified to include these features.
An example of a commonly used plasmid cloning vector is pUC19 (the lower case p denotes a plasmid), which is derived from a natural E. coli plasmid. The plasmid pUC19 is 2,686 bp long, contains an origin of replication that enables it to replicate in E. coli, and has a high copy number, which is useful when a large number of copies of the target DNA or its encoded protein are required (Fig. 2.5A). It has been genetically engineered to possess a short (54-bp) DNA sequence that contains many unique restriction endonuclease sites which is called a multiple-cloning site (also known as a polylinker) (Fig. 2.5B). A DNA sequence from the lactose operon of E. coli has also been added that includes a segment of the β-galactosidase gene (lacZ′) under the control of the lac promoter and a lacI gene that produces a repressor protein that regulates the expression of the lacZ′ gene from the lac promoter (Fig. 2.5A). The multiple-cloning site has been inserted within the β-galactosidase gene in a manner that does not disrupt the function of the β-galactosidase enzyme when it is expressed
